Multispecific binding constructs against checkpoint molecules and uses thereof

ABSTRACT

The present disclosure relates to compositions and methods for inhibiting tumor evasion by reducing immune checkpoint suppression. In some embodiments, provided herein are compositions that block the interaction between PD-1 and its ligand (e.g., PD-1 and/or PD-L2) while promoting the interaction of the cells on which PD-1 and its ligand are expressed. Also provided are methods of using such compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)of U.S. Provisional Application No. 62/760,801, filed on Nov. 13, 2018;U.S. Provisional Application No. 62/855,580, filed on May 31, 2019; U.S.Application No. 62/898,991, filed on Sep. 11, 2019; and U.S. ApplicationNo. 62/931,478, filed on Nov. 6, 2019. The foregoing applications areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a sequence listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 26, 2019, isnamed 1848492-0002-007-101_SL.txt and is 122,235 bytes in size.

BACKGROUND

Cancer is one of the leading causes of death in both the United Statesand worldwide. While common treatments such as surgery, radiation,chemotherapy, hormone therapy, targeted therapies, and immunotherapyhave decreased the rates of cancer-related deaths during the 20^(th)century, there were 14.1 million new cancer cases diagnosed and 8.2million cancer deaths worldwide as of 2012. Even with improved overallcancer survival rates during the 20^(th) century, cancer is stillresponsible for one in seven deaths worldwide. See American CancerSociety, Global Cancer Facts & FIGS. 3^(rd) Edition, Atlanta: AmericanCancer Society; 2015.

In recent years, an increasing body of evidence suggests that the immunesystem operates as a significant barrier to tumor formation andprogression. The principle that naturally-occurring T cells withanti-tumor potential or activity exist in a patient with cancer hasrationalized the development of immunotherapeutic approaches inoncology. Immune cells, such as T cells, macrophages, and natural killercells, can exhibit anti-tumor activity and effectively control theoccurrence and growth of malignant tumors. Tumor-specific or -associatedantigens can induce immune cells to recognize and eliminate malignancies(Chen & Mellman, (2013) Immunity 39(1):1-10). In spite of the existenceof tumor-specific immune responses, malignant tumors often evade oravoid immune attack through a variety of immunomodulatory mechanismsresulting in the failure to control tumor occurrence and progression(Motz & Coukos, (2013) Immunity 39(1):61-730). Indeed, an emerginghallmark of cancer is the exploitation of these immunomodulatorymechanisms and the disablement of anti-tumor immune responses, resultingin tumor evasion and escape from immunological killing (Hanahan andWeinberg (2011) Cell 144(5):646-674).

Novel approaches in the immunotherapy of cancer involve counteractingthese immune evasion and escape mechanisms and inducing the endogenousimmune system to reject tumors. However, there remains a need for noveltherapeutics that effectively counteract immune evasion, particularly incancer therapeutics.

SUMMARY OF THE DISCLOSURE

The present disclosure is based, in part, on novel multispecific andmultivalent constructs targeting both PD-1 and PD-L1, such as abispecific and tetravalent construct. As demonstrated herein, thesemultispecific constructs have improved in vitro and in vivo potency ascompared to combinations of individual antibodies, as well as comparedto clinical checkpoint blockade agents. Also provided herein are novelmonoclonal anti-PD-1 antibodies and antigen-binding fragments thereof,and novel monoclonal anti-PD-L1 antibodies and antigen-binding fragmentsthereof, for use in such multispecific and multivalent constructs. Someof these novel monoclonal anti-PD-1 antibodies and novel monoclonalanti-PD-L1 antibodies share a common light chain, thereby allowing forthe generation of multispecific and multivalent constructs havingsurprising excellent drug-like properties (DLPs) and ease ofmanufacturability, as well as affinities similar to their parentalantibodies. The present disclosure is also based, in part, on thediscovery that blocking the interaction between PD-1 expressed by animmune cell and its ligand (e.g., PD-L1 or PD-L2) expressed on a secondcell, while bridging the immune cell and the second cell (e.g., anotherimmune cell, or a tumor cell that expresses a PD-1 ligand) stronglyenhance, for example, T cell proliferation, IFNγ production andsecretion, and the cytolytic activity of T cells. Thus, provided hereinare compositions that block the interaction between PD-1 and its ligandwhile promoting the interaction of (bridging) the cells on which PD-1and its ligand (PD-L1 or PD-L2) are expressed. As exemplified herein,such compositions of the present disclosure with the capacity to “blockand bridge” provide superior anti-tumor efficacy (as measured, e.g., byIFNγ production and secretion and in vivo activity) as compared to,e.g., a cocktail having a stoichiometric amount of agents thatseparately bind the receptor and ligand; or a single agent that bindseither the receptor or the ligand. It was also found that themultispecific and multivalent constructs targeting both PD-1 and PD-L1described herein cause loss of expression of PD-1 on the cell surface,in a valency-dependent fashion. This loss of PD-1 expression was notobserved when a combination of the parental antibodies were used instoichiometrically equivalent amounts. Accordingly, the multispecificand multivalent constructs targeting both PD-1 and PD-L1 describedherein provide novel immunotherapeutic agents with increased potency andefficacy for use in the treatment of cancer.

In some embodiments, any of the multispecific antigen-binding constructsdisclosed herein binds to at least two different receptors or epitopes(e.g., PD-1 and PD-L1), wherein the two different receptors or epitopesbound by the multispecific antigen-binding construct are expressed onthe surface of the same cell. For example, in some embodiments, themultispecific antigen-binding construct simultaneously binds to PD-1 andPD-L1, wherein the PD-1 and PD-L1 are expressed on the surface of thesame cell. In some embodiments, any of the multispecific antigen-bindingconstructs disclosed herein binds to at least two different receptors orepitopes (e.g., PD-1 and PD-L1), wherein the two different receptors orepitopes bound by the multispecific antigen-binding construct areexpressed on the surface of two different cells. For example, in someembodiments, the multispecific antigen-binding construct simultaneouslybinds to PD-1 expressed on the surface of a first cell and to a PD-1ligand, e.g., PD-L1 or PD-L2, expressed on the surface of a second cell.

In some embodiments, the disclosure provides for a multispecificantigen-binding construct comprising at least two antigen-binding arms,wherein a first arm binds PD-1 expressed by an immune cell, and a secondarm binds a PD-1 ligand which is expressed by a second cell, wherein themultispecific antigen-binding construct blocks the interaction of PD-1and PD-1 ligand. In some embodiments, the PD-1 ligand is PD-L2. In someembodiments, the PD-1 ligand is PD-L1. In some embodiments, the immunecell is a T cell. In some embodiments, the T cell is a CD8+ T cell. Insome embodiments, the immune cell is a natural killer (NK) cell. In someembodiments, the immune cell is a macrophage. In some embodiments, thesecond cell is a second immune cell. In some embodiments, the secondimmune cell is any one or more of a T cell, a B cell, a macrophage, amyeloid-derived suppressor cell, a dendritic cell, or a mesenchymalstromal cell. In some embodiments, the second immune cell is aregulatory T cell. In some embodiments, the second cell is a tumor cell.In some embodiments, the tumor cell is selected from the groupconsisting of a hematological cancer, a lymphoma, a myeloma, a leukemia,a neurological cancer, melanoma, breast cancer, a prostate cancer, acolorectal cancer, lung cancer, head and neck cancer, a gastrointestinalcancer, liver cancer, pancreatic cancer, a genitourinary cancer, a bonecancer, renal cancer, and a vascular cancer. In some embodiments, botharms have a K_(D) of at least 1×10⁻⁷ M, at least 1×10⁻⁸M, at least1×10⁻⁹M, or at least 1×10⁻¹⁰ M. In some embodiments, the binding of onearm to its target does not block the binding of the other arm to itstarget. In some embodiments, the first arm and second arm bind to theirrespective targets and both arms remain bound concurrently. In someembodiments, binding of the first arm and the second arm to theirrespective targets can bridge the immune cell and the second celltogether. In some embodiments, the bridging of the immune cell and thesecond cell is determined by flow cytometry. In some embodiments, thefirst arm is an antagonist of PD-1.

In some embodiments of the multispecific antigen-binding constructs, thefirst arm binds to PD-1 and comprises: (a) a heavy chain variable regioncomprising (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX₁X₂YAX₃X₄, whereinX₁=S, R, G, or N; X₂=D, S, N, A, R, or G; X₃=M or L; X₄=S, L, or N);(ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a CDRH3comprising SEQ ID NO: 72 (ARGLDFIVGX₅TGNDY, wherein X₅=A, Y, or R); and(b) a light chain variable region comprising: (i) a CDRL1 comprising SEQID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS);and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the first arm comprises SEQ ID NO: 73(FTFSDYAMS), CDRH2 of the first arm comprises SEQ ID NO: 71(SAISNSGTYTYYA) and CDRH3 of the first arm comprises SEQ ID NO: 74(ARGLDFIVGATGNDY). In some embodiments, the CDRH1 of the first armcomprises SEQ ID NO: 73 (FTFSDYAMS), CDRH2 of the first arm comprisesSEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the first arm comprises SEQID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, the CDRH1 of the firstarm comprises SEQ ID NO: 76 (FTFSSYAMS), CDRH2 of the first armcomprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the first armcomprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 77 (FTFSSYAML), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 78 (FTFSNYALS), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 79 (FTFSAYAMN), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 81 (FTFGRYAMS), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 82 (FTFNSYAMS), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 75 (ARGLDFIVGYTGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 83 (FTFSNYAMS), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 74 (ARGLDFIVGATGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 84 (FTFSGYAMS), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 86 (FTFSSYAMN), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY). In some embodiments, theCDRH1 of the first arm comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 of thefirst arm comprises SEQ ID NO: 71 (SAISNSGTYTYYA) and CDRH3 of the firstarm comprises SEQ ID NO: 85 (ARGLDFIVGRTGNDY).

In some embodiments, the CDRL1 of the first arm comprises SEQ ID NO: 9(RASQSISSYLN), CDRL2 of the first arm comprises SEQ ID NO: 5 (AASSLQS)and CDRL3 of the first arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the heavy chain variable region of the first armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 87. In some embodiments, the heavy chain variable region of thefirst arm comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 88. In some embodiments, the heavy chainvariable region of the first arm comprises an amino acid sequence thatis at least 90% identical to SEQ ID NO: 89. In some embodiments, theheavy chain variable region of the first arm comprises an amino acidsequence that is at least 90% identical to SEQ ID NO: 90. In someembodiments, the heavy chain variable region of the first arm comprisesan amino acid sequence that is at least 90% identical to SEQ ID NO: 91.In some embodiments, the heavy chain variable region of the first armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 92. In some embodiments, the heavy chain variable region of thefirst arm comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 93. In some embodiments, the heavy chainvariable region of the first arm comprises an amino acid sequence thatis at least 90% identical to SEQ ID NO: 94. In some embodiments, theheavy chain variable region of the first arm comprises an amino acidsequence that is at least 90% identical to SEQ ID NO: 95. In someembodiments, the heavy chain variable region of the first arm comprisesan amino acid sequence that is at least 90% identical to SEQ ID NO: 96.In some embodiments, the heavy chain variable region of the first armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 97. In some embodiments, the heavy chain variable region of thefirst arm comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 98. In some embodiments, the heavy chainvariable region of the first arm comprises an amino acid sequence thatis at least 90% identical to SEQ ID NO: 99. In some embodiments, thelight chain variable region of the first arm comprises an amino acidsequence that is at least 90% identical to SEQ ID NO: 59.

In some embodiments, the second arm is an antagonist of the PD-1 ligand.In some embodiments, the second arm is an antagonist of PD-L2. In someembodiments, the second arm is an antagonist of PD-L1. In someembodiments, the second arm binds to PD-L1 and comprises: a. a heavychain variable region comprising (i) a CDRH1 comprising SEQ ID NO: 1(GTFSSYAIN); (ii) a CDRH2 comprising SEQ ID NO: 2 (GGIIPX₁X₂GX₃ATYA,wherein X₁ is V or I; X₂ is F, L, or V; and X₃ is T or A); and (iii) aCDRH3 comprising SEQ ID NO: 3 (ARLKX₁ELKDAFDI, wherein X₁ is G, F, orN); and b. a light chain variable region comprising: (i) a CDRL1comprising SEQ ID NO: 4 (RASQX₁ISSYLN, wherein X₁ is S, W, or Q); (ii) aCDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprisingSEQ ID NO: 6 (X₁QSYSTPLT, wherein X₁ is Q or F).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 7(GGIIPILGAATYA) and CDRH3 of the second arm comprises SEQ ID NO: 8(ARLKGELKDAFDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 7 (GGIIPILGAATYA), CDRH3 of the second arm comprises SEQ IDNO: 8 (ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11(GGIIPVFGTATYA), CDRH3 of the second arm comprises SEQ ID NO: 8(ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT). In someembodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11(GGIIPVFGTATYA) and CDRH3 of the second arm comprises SEQ ID NO: 8(ARLKGELKDAFDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 of the second arm comprises SEQ IDNO: 8 (ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 12(RASQWISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT). In someembodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11(GGIIPVFGTATYA), CDRH3 of the second arm comprises SEQ ID NO: 8(ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 13(RASQQISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT). In someembodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11(GGIIPVFGTATYA), CDRH3 of the second arm comprises SEQ ID NO: 8(ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 15(GGIIPIFGIANYA) and CDRH3 of the second arm comprises SEQ ID NO: 8(ARLKGELKDAFDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 15 (GGIIPIFGIANYA), CDRH3 of the second arm comprises SEQ IDNO: 8 (ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 16(GGIIPNFGTATYA) and CDRH3 of the second arm comprises SEQ ID NO: 17(ARLKGELKGAGDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 16 (GGIIPNFGTATYA), CDRH3 of the second arm comprises SEQ IDNO: 17 (ARLKGELKGAGDI); CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11(GGIIPVFGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 18(ARLKFELKDAFDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 of the second arm comprises SEQ IDNO: 18 (ARLKFELKDAFDI), CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11(GGIIPVFGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 19(ARLKGELKDAFDE). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 of the second arm comprises SEQ IDNO: 19 (ARLKGELKDAFDE), CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11(GGIIPVFGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 20(ARLKNELKDAFDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 of the second arm comprises SEQ IDNO: 20 (ARLKNELKDAFDI), CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 21(GGVIPFLGTANYA), and CDRH3 of the second arm comprises SEQ ID NO: 22(ARLKGILKDALDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 21 (GGVIPFLGTANYA), CDRH3 of the second arm comprises SEQ IDNO: 22 (ARLKGILKDALDI), CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 29(GRIIPIFGTADYA), and CDRH3 of the second arm comprises SEQ ID NO: 8(ARLKGELKDAFDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 29 (GRIIPIFGTADYA), CDRH3 of the second arm comprises SEQ IDNO: 8 (ARLKGELKDAFDI), CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 31(GGIIPILGTATYA), and CDRH3 of the second arm comprises SEQ ID NO: 32(ARRKGELKDAFDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 31 (GGIIPILGTATYA), CDRH3 of the second arm comprises SEQ IDNO: 32 (ARRKGELKDAFDI), CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 33(GGIIPIVATANYA), and CDRH3 of the second arm comprises SEQ ID NO: 32(ARRKGELKDAFDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 33 (GGIIPIVATANYA), CDRH3 of the second arm comprises SEQ IDNO: 32 (ARRKGELKDAFDI), CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 34(GGIIPIFGKATYA), and CDRH3 of the second arm comprises SEQ ID NO: 32(ARRKGELKDAFDI). In some embodiments, the CDRH1 of the second armcomprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 of the second arm comprisesSEQ ID NO: 34 (GGIIPIFGKATYA), CDRH3 of the second arm comprises SEQ IDNO: 32 (ARRKGELKDAFDI), CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11(GGIIPVFGTATYA), CDRH3 of the second arm comprises SEQ ID NO: 8(ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 38 (FQSYSTPLT).

In some embodiments, the CDRH1 of the second arm comprises SEQ ID NO: 1(GTFSSYAIN), CDRH2 of the second arm comprises SEQ ID NO: 11(GGIIPVFGTATYA), CDRH3 of the second arm comprises SEQ ID NO: 8(ARLKGELKDAFDI); CDRL1 of the second arm comprises SEQ ID NO: 9(RASQSISSYLN); CDRL2 of the second arm comprises SEQ ID NO: 5 (AASSLQS);and CDRL3 of the second arm comprises SEQ ID NO: 39 (QQSYSTILT).

In some embodiments, the second arm comprises: a. a heavy chain variableregion comprising (i) a CDRH1 comprising SEQ ID NO: 14 (GTFSSYAFS), (ii)a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA) and (iii) a CDRH3comprising SEQ ID NO: 8 (ARLKGELKDAFDI); and b. a light chain variableregion comprising: (i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN);(ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3comprising SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the second arm comprises a heavy chain variableregion comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii)a CDRH2 comprising SEQ ID NO: 24 (GGIIPIVGIANYA), and (iii) a CDRH3comprising SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments, the secondarm comprises a light chain variable region comprising: (i) a CDRL1comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ IDNO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the second arm comprises a heavy chain variableregion comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii)a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) a CDRH3comprising SEQ ID NO: 25 (ARLKGEFKDAFDI). In some embodiments, thesecond arm comprises a light chain variable region comprising: (i) aCDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10(QQSYSTPLT).

In some embodiments, the second arm comprises a heavy chain variableregion comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii)a CDRH2 comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and (iii) a CDRH3comprising SEQ ID NO: 8 (ARLKGELKDAFDI). In some embodiments, the secondarm comprises a light chain variable region comprising: (i) a CDRL1comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ IDNO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments, the second arm comprises a heavy chain variableregion comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii)a CDRH2 comprising SEQ ID NO: 27 (GRINPILGTANYA), and (iii) a CDRH3comprising SEQ ID NO: 28 (ARLKGELKDAFSI). In some embodiments, thesecond arm comprises a light chain variable region comprising: (i) aCDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10(QQSYSTPLT).

In some embodiments, the second arm comprises a heavy chain variableregion comprising (i) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), (ii)a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) a CDRH3comprising SEQ ID NO: 30 (ARLKGELKCAFDI). In some embodiments, thesecond arm comprises a light chain variable region comprising: (i) aCDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10(QQSYSTPLT).

In some embodiments, the second arm comprises a heavy chain variableregion comprising (i) a CDRH1 comprising SEQ ID NO: 122 (GTKSSYAIS),(ii) CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) a CDRH3comprising SEQ ID NO: 30 (ARLKGELKCAFDI). In some embodiments, thesecond arm further comprises a light chain variable region comprising:(i) a CDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2comprising SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ IDNO: 10 (QQSYSTPLT).

In some embodiments, the second arm comprises a heavy chain variableregion comprising (i) a CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), (ii)a CDRH2 comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and (iii) a CDRH3comprising SEQ ID NO: 37 (ARLKSELKDAFDI). In some embodiments, thesecond arm comprises a light chain variable region comprising: (i) aCDRL1 comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 10(QQSYSTPLT).

In some embodiments, the second arm that binds PD-L1 comprises a heavychain variable region comprising an amino acid sequence that is at least90% identical to any one of SEQ ID NOs: 35, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58 and a light chainvariable region comprising an amino acid sequence that is at least 90%identical to any one of SEQ ID NOs: 59, 60, 61, 62, or 63.

In some embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 35 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTMV TVSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 40 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVT VSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 41 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVT VSS). In someembodiments, the heavy chain variable region of the second arm comprisesan amino acid sequence that is at least 90% identical to SEQ ID NO: 42(QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPNFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKGAGDIWGQGTLV TVSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 43 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKFELKDAFDIWGQGTLVT VSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 44 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDEWGQGTLV TVSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 45 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVT AST). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 46 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKNELKDAFDIWGQGTLVT VSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 47 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGVIPFLGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGILKDALDIWGQGTLV TVSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 48 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQDLEWMGGIIPIVGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVT VSS). In someembodiments, the heavy chain variable region of the second arm comprisesan amino acid sequence that is at least 90% identical to SEQ ID NO: 49(QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGEFKDAFDIWGQGTLVT VSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 50 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPLFGTAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLV TVSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 51 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRINPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFSIWGQGTLVT VSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 52 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGRIIPIFGTADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLV TVSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 53 (QVQLVQSGAEVKKPGSSVKVSCKASGGKFSSYAISWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKCAFDIWGQGTLVT VSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 54 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVT VSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 55 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGAATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVT VSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 56 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIVATANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLV TVSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 57 (QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGKATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVT VSS). Insome embodiments, the heavy chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 58 (QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSHAVSWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKSELKDAFDIWGQGTLV TVSS). Insome embodiments, the light chain variable region of the second armcomprises an amino acid sequence that is at least 90% identical to SEQID NO: 59 (DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK). In some embodiments,the light chain variable region of the second arm comprises an aminoacid sequence that is at least 90% identical to SEQ ID NO: 60(DIQMTQSPSSLSASVGDRVTITCRASQWISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK). In some embodiments,the light chain variable region of the second arm comprises an aminoacid sequence that is at least 90% identical to SEQ ID NO: 61(DIQMTQSPSSLSASVGDRVTITCRASQQISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK). In some embodiments,the light chain variable region of the second arm comprises an aminoacid sequence that is at least 90% identical to SEQ ID NO: 62(DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQSYSTPLTFGGGTKVEIK). In some embodiments,the light chain variable region of the second arm comprises an aminoacid sequence that is at least 90% identical to SEQ ID NO: 63(DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTILTFGGGTKVEIK).

In some embodiments of any of the aspects described herein, theconstruct is a bispecific antibody. In some embodiments, the bispecificantibody is an antagonist of both PD-1 and PD-1 ligand. In someembodiments, the construct comprises a common light chain. In someembodiments, one or both of the arms is an aptamer. In some embodiments,one or both of the arms is a protein other than an antibody. In someembodiments, the construct comprises at least two bispecific antibodies.In some embodiments, one of the at least two bispecific antibodiesismonovalent for PD-1. In some embodiments, one of the at least twobispecific antibodies ismonovalent for PD-1 ligand. In some embodiments,at least one of the arms is a bivalent antibody specific for PD-1. Insome embodiments, at least one of the arms is a bivalent antibodyspecific for PD-L1. In some embodiments, at least one of the arms is abivalent antibody specific for PD-1, and at least one of the arms is abivalent antibody specific for PD-L1. In some embodiments, at least oneof the arms is bivalent for PD-1. In some embodiments, at least one ofthe arms is bivalent for PD-L1. In some embodiments, at least one of thearms is bivalent for PD-1, and at least one of the arms is bivalent forPD-L1. In some embodiments, the bispecific antibody binds two differentepitopes on PD-1. In some embodiments, the bispecific antibody binds twodifferent epitopes on the PD-1 ligand.

In some embodiments, any of the multispecific antigen-binding constructsdisclosed herein comprises at least two monospecific antibodies. In someembodiments, at least one of the monospecific antibodies is an anti-PD-1antibody. In some embodiments, the anti-PD-1 antibody is a bivalentanti-PD-1 antibody. In some embodiments, at least one of themonospecific antibodies is an anti-PD-L1 antibody. In some embodiments,the anti-PD-1 antibody is a bivalent anti-PD-L1 antibody. In someembodiments, the construct comprises a bivalent anti-PD-1 antibody and abivalent anti-PD-L1 antibody. In some embodiments, the construct is afusion construct in which a polypeptide comprising the variable heavychain of the anti-PD-1 antibody is fused to a polypeptide comprising thevariable heavy chain of the anti-PD-L1 antibody. In some embodiments,the polypeptide comprising the variable heavy chain of the anti-PD-1antibody is fused to the polypeptide comprising the variable heavy chainof the anti-PD-L1 antibody by means of a linker. In some embodiments,the fusion construct comprises a common light chain. In someembodiments, the N-terminal variable heavy chain of the fusion constructbinds to PD-1 in the presence of the common light chain, and theC-terminal variable heavy chain of the fusion construct binds to PD-L1in the presence of the common light chain. In some embodiments, theN-terminal variable heavy chain of the fusion construct binds to PD-L1in the presence of the common light chain, and the C-terminal variableheavy chain of the fusion construct binds to PD-1 in the presence of thecommon light chain.

In some aspects and embodiments, the disclosure provides for amultispecific antigen-binding construct comprising at least two units ofantigen-binding, wherein a first unit of antigen-binding binds PD-1, anda second unit of antigen-binding binds a PD-1 ligand. In someembodiments, the first unit of antigen-binding binds PD-1 expressed byan immune cell. In some embodiments, the second unit of antigen-bindingbinds PD-1 expressed by a second cell. In some embodiments, themultispecific antigen-binding construct blocks the interaction of PD-1and a PD-1 ligand, such as PD-L1 or PD-L2. In some embodiments, themultispecific antigen-binding construct blocks the interaction of PD-1and a PD-1 ligand, such as PD-L1 or PD-L2. In some embodiments, themultispecific antigen-binding construct comprises at least two units ofantigen-binding that bind PD-1. In some embodiments, the multispecificantigen-binding construct comprises two units of antigen-binding thatbind PD-1. In some embodiments, the multispecific antigen-bindingconstruct comprises at least two units of antigen-binding that bind aPD-1 ligand, such as PD-L1 or PD-L2. In some embodiments, themultispecific antigen-binding construct comprises two units ofantigen-binding that bind a PD-1 ligand, such as PD-L1 or PD-L2. In someembodiments, the multispecific antigen-binding construct comprises atleast four units of antigen-binding, wherein two units ofantigen-binding bind PD-1 and two units of antigen-binding bind a PD-1ligand, such as PD-L1 or PD-L2. In some embodiments, the multispecificantigen-binding construct comprises four units of antigen-binding,wherein two units of antigen-binding bind PD-1 and two units ofantigen-binding bind a PD-1 ligand, such as PD-L1 or PD-L2. In someembodiments, each unit of antigen-binding is capable of bindingindependently to its cognate antigen, i.e., PD-1 or a PD-1 ligand, suchas PD-L1 or PD-L2. In some embodiments, the multispecificantigen-binding construct promotes loss of PD-1 expression from a cell.In some embodiments, the loss of PD-1 expression is due to PD-1shedding. In some embodiments, the multispecific antigen-bindingconstruct blocks interaction of PD-1 and the PD-1 ligand, such as PD-L1or PD-L2. In some embodiments, the multispecific antigen-bindingconstruct comprises a common light chain. For example, at least twounits of antigen-binding comprise a common light chain.

In some embodiments, the first unit of antigen-binding binds PD-1 andcomprises:

-   -   (a) a heavy chain variable region comprising (i) a CDRH1        comprising SEQ ID NO: 70 (FTFX₁X₂YAX₃X₄, wherein X₁=S, R, G, or        N; X₂=D, S, N, A, R, or G; X₃=M or L; X₄=S, L, or N); (ii) a        CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a        CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX₅TGNDY, wherein X₅=A,        Y, or R); and    -   (b) a light chain variable region comprising: (i) a CDRL1        comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising        SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO:        10 (QQSYSTPLT).

In some such embodiments, the first unit of antigen-binding binds PD-1and comprises:

-   -   (a) a CDRH1 comprising SEQ ID NO: 73 (FTFSDYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 74 (ARGLDFIVGATGNDY);    -   (b) a CDRH1 comprising SEQ ID NO: 73 (FTFSDYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (c) a CDRH1 comprising SEQ ID NO: 76 (FTFSSYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (d) a CDRH1 comprising SEQ ID NO: 77 (FTFSSYAML), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (e) a CDRH1 comprising SEQ ID NO: 78 (FTFSNYALS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (f) a CDRH1 comprising SEQ ID NO: 79 (FTFSAYAMN), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (g) a CDRH1 comprising SEQ ID NO: 80 (FTFRSYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (h) a CDRH1 comprising SEQ ID NO: 81 (FTFGRYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (i) a CDRH1 comprising SEQ ID NO: 82 (FTFNSYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (j) a CDRH1 comprising SEQ ID NO: 83 (FTFSNYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 74 (ARGLDFIVGATGNDY);    -   (k) a CDRH1 comprising SEQ ID NO: 84 (FTFSGYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 85 (ARGLDFIVGRTGNDY);    -   (l) a CDRH1 comprising SEQ ID NO: 86 (FTFSSYAMN), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA) and a CDRH3 comprising        SEQ ID NO: 85 (ARGLDFIVGRTGNDY); or    -   (m) a CDRH1 comprising SEQ ID NO: 80 (FTFRSYAMS), a comprising        SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising SEQ ID NO:        85 (ARGLDFIVGRTGNDY).

In some embodiments, the first unit of antigen-binding binds PD-1 andcomprises:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 87;    -   (b) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 88;    -   (c) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 89;    -   (d) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 90;    -   (e) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 91;    -   (f) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 92;    -   (g) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 93;    -   (h) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 94;    -   (i) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 95;    -   (j) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 96;    -   (k) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 97;    -   (l) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 98; or    -   (m) a heavy chain variable region comprising amino acid sequence        that is at least 90% identical to SEQ ID NO: 99.

In some embodiments, the first unit of antigen-binding binds PD-1 andcomprises a light chain variable region comprising an amino acidsequence that is at least 90% identical to SEQ ID NO: 59.

In some embodiments, the second unit of antigen-binding binds PD-L2. Insome embodiments, the second unit of antigen-binding binds PD-L1. Insome embodiments, the second unit of antigen-binding binds PD-L1 andcomprises:

-   -   a. a heavy chain variable region comprising (i) a CDRH1        comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2 comprising SEQ        ID NO: 2 (GGIIPX₁X₂GX₃ATYA, wherein X₁ is V or I; X₂ is F, L, or        V; and X₃ is T or A); and (iii) a CDRH3 comprising SEQ ID NO: 3        (ARLKX₁ELKDAFDI, wherein X₁ is G, F, or N); and    -   b. a light chain variable region comprising: (i) a CDRL1        comprising SEQ ID NO: 4 (RASQX₁ISSYLN, wherein X₁ is S, W, or        Q); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a        CDRL3 comprising SEQ ID NO: 6 (X₁QSYSTPLT, wherein X₁ is Q or        F).

In some such embodiments, the second unit of antigen-binding binds PD-L1and comprises:

-   -   (a) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 7 (GGIIPILGAATYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (b) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 7 (GGIIPILGAATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (c) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (d) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA) and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (e) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 12        (RASQWISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (f) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 13        (RASQQISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (g) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (h) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 15 (GGIIPIFGIANYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 15 (GGIIPIFGIANYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (CQQSYSTPLTF);    -   (j) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 16 (GGIIPNFGTATYA), and a CDRH3 comprising        SEQ ID NO: 17 (ARLKGELKGAGDI);    -   (k) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 16 (GGIIPNFGTATYA), a CDRH3 comprising SEQ        ID NO: 17 (ARLKGELKGAGDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (l) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 18 (ARLKFELKDAFDI);    -   (m) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 18 (ARLKFELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (n) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 19 (ARLKGELKDAFDE);    -   (o) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 19 (ARLKGELKDAFDE), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (p) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 20 (ARLKNELKDAFDI);    -   (q) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 20 (ARLKNELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (r) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 21 (GGVIPFLGTANYA), and a CDRH3 comprising        SEQ ID NO: 22 (ARLKGILKDALDI);    -   (s) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 21 (GGVIPFLGTANYA), a CDRH3 comprising SEQ        ID NO: 22 (ARLKGILKDALDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (t) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising EQ ID NO: 29 (GRIIPIFGTADYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (u) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 29 (GRIIPIFGTADYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (v) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 31 (GGIIPILGTATYA), and a CDRH3 comprising        SEQ ID NO: 32 (ARRKGELKDAFDI);    -   (w) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 31 (GGIIPILGTATYA), a CDRH3 comprising SEQ        ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (x) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 33 (GGIIPIVATANYA), and a CDRH3 comprising        SEQ ID NO: 32 (ARRKGELKDAFDI);    -   (y) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 33 (GGIIPIVATANYA), a CDRH3 comprising SEQ        ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (z) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 34 (GGIIPIFGKATYA), and a CDRH3 comprising        SEQ ID NO: 32 (ARRKGELKDAFDI);    -   (aa) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 34 (GGIIPIFGKATYA), a CDRH3 comprising SEQ        ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (bb) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 38 (FQSYSTPLT);    -   (cc) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 39 (QQSYSTILT);    -   (dd) a CDRH1 comprising SEQ ID NO: 14 (GTFSSYAFS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA) and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (ee) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 24 (GGIIPIVGIANYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (ff) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 24 (GGIIPIVGIANYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (gg) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 25 (ARLKGEFKDAFDI);    -   (hh) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 25 (ARLKGEFKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (ii) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (jj) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (kk) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 27 (GRINPILGTANYA), and a CDRH3 comprising        SEQ ID NO: 28 (ARLKGELKDAFSI);    -   (ll) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 27 (GRINPILGTANYA), and a CDRH3 comprising        SEQ ID NO: 28 (ARLKGELKDAFSI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (mm) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 30 (ARLKGELKCAFDI);    -   (nn) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a comprising SEQ        ID NO: 30 (ARLKGELKCAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (oo) a CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 37 (ARLKSELKDAFDI); or    -   (pp) a CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 37 (ARLKSELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

In some such embodiments, the second unit of antigen-binding binds PD-L1and comprises and comprises a heavy chain variable region comprising anamino acid sequence that is at least 90% identical to any one of SEQ IDNOs: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, or 58, and a light chain variable region comprising an aminoacid sequence that is at least 90% identical to any one of SEQ ID NOs:59, 60, 61, 62, or 63.

In some embodiments, the second unit of antigen-binding binds PD-L1 andcomprises a heavy chain variable region comprising:

(a) an amino acid sequence that is at least 90% identical to SEQ ID NO:35;

(b) an amino acid sequence that is at least 90% identical to SEQ ID NO:40;

(c) an amino acid sequence that is at least 90% identical to SEQ ID NO:41;

(d) an amino acid sequence that is at least 90% identical to SEQ ID NO:42;

(e) an amino acid sequence that is at least 90% identical to SEQ ID NO:43;

(f) an amino acid sequence that is at least 90% identical to SEQ ID NO:44;

(g) an amino acid sequence that is at least 90% identical to SEQ ID NO:45;

(h) an amino acid sequence that is at least 90% identical to SEQ ID NO:46;

(i) an amino acid sequence that is at least 90% identical to SEQ ID NO:47;

(j) an amino acid sequence that is at least 90% identical to SEQ ID NO:48;

(k) an amino acid sequence that is at least 90% identical to SEQ ID NO:49;

(l) an amino acid sequence that is at least 90% identical to SEQ ID NO:50;

(m) an amino acid sequence that is at least 90% identical to SEQ ID NO:51;

(n) an amino acid sequence that is at least 90% identical to SEQ ID NO:52;

(o) an amino acid sequence that is at least 90% identical to SEQ ID NO:53;

(p) an amino acid sequence that is at least 90% identical to SEQ ID NO:54;

(q) an amino acid sequence that is at least 90% identical to SEQ ID NO:55;

(r) an amino acid sequence that is at least 90% identical to SEQ ID NO:56;

(s) an amino acid sequence that is at least 90% identical to SEQ ID NO:57; or

(t) an amino acid sequence that is at least 90% identical to SEQ ID NO:58;

In some embodiments, the second unit of antigen-binding binds PD-L1 andcomprises a light chain variable region comprising:

-   -   (a) an amino acid sequence that is at least 90% identical to SEQ        ID NO: 59;    -   (b) an amino acid sequence that is at least 90% identical to SEQ        ID NO: 60;    -   (c) an amino acid sequence that is at least 90% identical to SEQ        ID NO: 61;    -   (d) an amino acid sequence that is at least 90% identical to SEQ        ID NO: 62; or    -   (e) an amino acid sequence that is at least 90% identical to SEQ        ID NO: 63.

Also provided herein, in some aspects and embodiments, is amultispecific antigen-binding construct comprising four units ofantigen-binding, wherein two units of antigen-binding bind PD-1 and twounits of antigen-binding bind PD-L1, and wherein the construct comprisesa heavy chain amino acid sequence that is at least 85%, identical to theamino acid sequence of SEQ ID NO: 100 or 102, and a light chain aminoacid sequence that is at least 85%, identical to the amino acid sequenceof SEQ ID NO: 101 or 103.

Also provided herein, in some aspects and embodiments, is amultispecific antigen-binding construct comprising four units ofantigen-binding, wherein two units of antigen-binding bind PD-1 and twounits of antigen-binding bind PD-L1, and wherein the construct comprisesa heavy chain amino acid sequence that is at least 85%, identical to theamino acid sequence of SEQ ID NO: 100 and a light chain amino acidsequence that is at least 85%, identical to the amino acid sequence ofSEQ ID NO: 101.

Also provided herein, in some aspects and embodiments, is amultispecific antigen-binding construct comprising four units ofantigen-binding, wherein two units of antigen-binding bind PD-1 and twounits of antigen-binding bind PD-L1, and wherein the construct comprisesa heavy chain amino acid sequence that is at least 85%, identical to theamino acid sequence of SEQ ID NO: 102 and a light chain amino acidsequence that is at least 85%, identical to the amino acid sequence ofSEQ ID NO: 103.

In some embodiments, the construct does not comprise an Fc domain. Insome embodiments, the first arm or second arm, or both, comprises aheavy chain comprising one or more immunoglobulin Fc modifications. Insome embodiments, the immunoglobulin Fc domain of the heavy chaincomprises one or more amino acid mutations that promoteheterodimerization of the first and second arms. In some embodiments,the mutation is present in a CH3 domain of the heavy chain. In someembodiments, the multispecific antigen-binding construct is produced ina quadroma cell. In some embodiments, the construct comprises one ormore immunoglobulin constant region modifications. In some embodiments,the immunoglobulin constant region comprises one or more amino acidmutations that promote heterodimerization of antibodies. In someembodiments, one or more mutations is present in the light chainconstant region of one arm and one or more mutations is present in theheavy chain constant region of another arm. In some embodiments, thebispecific antibody is of a format selected from the group consisting ofa bispecific IgG, bispecific antibody fragment, bispecific fusionprotein, appended IgG, and bispecific antibody conjugate. In someembodiments, the Fc region has reduced effector function. In someembodiments, the Fc region enhances half-life of the construct.

In some embodiments, the construct comprises a heavy chain amino acidsequence that is at least 85%, identical to the amino acid sequence ofSEQ ID NO: 100 or 102. In some embodiments, the construct comprises alight chain amino acid sequence that is at least 85%, identical to theamino acid sequence of SEQ ID NO: 101 or 103. In some embodiments, theconstruct comprises a heavy chain amino acid sequence that is at least85% identical to the amino acid sequence of SEQ ID NO: 100, and whereinthe construct comprises a light chain amino acid sequence that is atleast 85% identical to the amino acid sequence of SEQ ID NO: 101. Insome embodiments, the construct comprises a heavy chain amino acidsequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 102, and wherein the construct comprises a light chain aminoacid sequence that is at least 85% identical to the amino acid sequenceof SEQ ID NO: 103.

In some embodiments, any of the multispecific antigen-binding constructsdisclosed herein are aglycosylated. In some embodiments, themultispecific antigen-binding construct is capable of binding humanPD-1. In some embodiments, the multispecific antigen-binding constructis capable of binding murine PD-1. In some embodiments, themultispecific antigen-binding construct is capable of binding cynomolgusmonkey PD-1. In some embodiments, the multispecific antigen-bindingconstruct is capable of binding human, murine and cynomolgus monkey PD-1with similar affinity.

In some embodiments, any of the multispecific antigen-binding constructsdisclosed herein is capable of reducing PD-1 levels on a cell. In someembodiments, the multispecific antigen-binding construct is capable ofinducing PD-1 degradation. In some embodiments, the multispecificantigen-binding construct is capable of reducing PD-1 expression. Insome embodiments, the multispecific antigen-binding construct is capableof reducing PD-1 cell surface expression. In some embodiments, themultispecific antigen-binding construct is capable of reducing PD-1 cellsurface expression by inducing shedding of PD-1 from the cell surface.In some embodiments, the multispecific antigen-binding construct iscapable of binding both PD-1 and PD-L1 and reducing PD-1 levels on acell. In some embodiments, the multispecific antigen-binding constructis capable of binding both PD-1 and PD-L1 and inducing PD-1 degradation.In some embodiments, the multispecific antigen-binding construct iscapable of binding both PD-1 and PD-L1 and reducing PD-1 expression. Insome embodiments, the multispecific antigen-binding construct is capableof inducing shedding of PD-1 from an immune cell. In some embodiments,the multispecific antigen-binding construct is capable of binding bothPD-1 and PD-L1 and inducing PD-1 shedding from an immune cell. In someembodiments, the multispecific antigen-binding construct is capable ofsequestering PD-L1, such that PD-L1 cannot bind CD80. In someembodiments, the multispecific antigen-binding construct is capable ofsequestering PD-L1, such that PD-L1 cannot bind CD80, and wherein CD80is free to bind CD28. In some embodiments, the cell is an immune cell,such as a T cell. In some embodiments, the immune cell (e.g., T cell) isa tumor infiltrating lymphocyte (TIL). In some embodiments, engagementof a multispecific antigen-binding molecule described herein to PD-1expressed by an immune cell in the tumor microenvironment results in thedownregulation of PD-1 by the immune cell. In some embodiments, theimmune cell is a T cell. In some embodiments, the immune cell (e.g., Tcell) is a tumor infiltrating lymphocyte (TIL).

In some embodiments, any of the multispecific antigen-binding constructsdisclosed herein is capable of inducing at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or99% greater interferon-gamma levels (e.g., as measured in aStaphylococcus aureus Enterotoxin A (“SEA”) assay) as compared to areference antigen-binding construct (e.g., pembrolizumab oratezolizumab) or to a reference combination of antigen-bindingconstructs (e.g., a composition comprising the PD-1 and PD-L1 arms ofthe multispecific antigen-binding construct, wherein the PD-1 and PD-L1arms in the composition are not conjugated to one another). In someembodiments, the multispecific antigen-binding construct is capable ofinducing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% greater interleukin-2levels (e.g., as measured in a SEA assay) as compared to a referenceantigen-binding construct (e.g., pembrolizumab or atezolizumab) or to areference combination of antigen-binding constructs (e.g., a compositioncomprising the PD-1 and PD-L1 arms of the multispecific antigen-bindingconstruct, wherein the PD-1 and PD-L1 arms in the composition are notconjugated to one another).

In some embodiments, the multispecific antigen-binding construct inducesat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99%, 200%, 300%, 400%, or 500% morekilling of tumor cells (e.g., leukemia cells, lymphoma cells, melanoma,or breast cancer cells), as compared to a reference antigen-bindingconstruct (e.g., pembrolizumab or atezolizumab) or to a referencecombination of antigen-binding constructs (e.g., a compositioncomprising the PD-1 and PD-L1 arms of the multispecific antigen-bindingconstruct, wherein the PD-1 and PD-L1 arms in the composition are notconjugated to one another).

In some embodiments, the multispecific antigen-binding construct iscapable of extending the survival of a subject suffering from a cancer(e.g., leukemia, lymphoma, melanoma, and/or breast cancer) by at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% longerthan a subject administered a reference antigen-binding construct (e.g.,pembrolizumab or atezolizumab) or a reference combination ofantigen-binding constructs (e.g., a composition comprising the PD-1 andPD-L1 arms of the multispecific antigen-binding construct, wherein thePD-1 and PD-L1 arms in the composition are not conjugated to oneanother). In some embodiments, the multispecific antigen-bindingconstruct is capable of inducing at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%,100%, 200%, 300%, 400%, or 500% more shedding of PD-1 from an immunecell as compared to an untreated immune cell or as compared to an immunecell treated with reference antigen-binding construct (e.g.,pembrolizumab or atezolizumab) or a reference combination ofantigen-binding constructs (e.g., a composition comprising the PD-1 andPD-L1 arms of the multispecific antigen-binding construct, wherein thePD-1 and PD-L1 arms in the composition are not conjugated to oneanother). In some embodiments, the multispecific antigen-bindingconstruct is capable of reducing PD-1 levels at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99%, 100%, 200%, 300%, 400%, or 500% (e.g., by causing PD-1shedding from the cell surface and/or inducing PD-1 degradation and/orreducing PD-1 expression) as compared to an untreated immune cell or toan immune cell treated with a reference antigen-binding construct (e.g.,pembrolizumab or atezolizumab) or a reference combination ofantigen-binding constructs (e.g., a composition comprising the PD-1 andPD-L1 arms of the multispecific antigen-binding construct, wherein thePD-1 and PD-L1 arms in the composition are not conjugated to oneanother).

In some embodiments, engagement of a multispecific antigen-bindingmolecule described herein to PD-1 expressed by a cell results in thedownregulation and/or loss of cell-surface expression of PD-1 by thecell. Such down-regulation or loss of cell-surface expression can be dueto, in part, for example, shedding of extracellular PD-1 from thesurface of the immune cell. In some embodiments, the cell is an immunecell, such as a T cell. In some embodiments, the immune cell (e.g., Tcell) is a tumor infiltrating lymphocyte (TIL). In some embodiments,engagement of a multispecific antigen-binding molecule described hereinto PD-1 expressed by an immune cell in the tumor microenvironmentresults in the downregulation of PD-1 by the immune cell. In someembodiments, the immune cell is a T cell. In some embodiments, theimmune cell (e.g., T cell) is a tumor infiltrating lymphocyte (TIL).

In some aspects, the disclosure provides for a method for treating aproliferative disorder in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of anyof the multispecific antigen-binding constructs disclosed herein,thereby treating the proliferative disorder in the subject. In someembodiments, the proliferative disorder is cancer. In some embodiments,the cancer is selected from the group consisting of a hematologicalcancer, a neurological cancer, melanoma, breast cancer, lung cancer,head and neck cancer, a gastrointestinal cancer, liver cancer,pancreatic cancer, a genitourinary cancer, a bone cancer, and a vascularcancer. In some embodiments, the disclosure provides for a method ofenhancing an immune response in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of anyof the multispecific antigen-binding constructs disclosed herein,thereby enhancing the immune response in the subject. In someembodiments, the enhanced immune response includes any one or more ofenhanced T cell function, enhanced NK cell function, or enhancedmacrophage function. In some embodiments, the enhancement of T cellfunction is greater upon administration of the multispecificantigen-binding construct, as compared to an agent that binds eitherPD-1 or a PD-1 ligand, or a cocktail comprising an agent that binds PD-1and an agent that binds a PD-1 ligand. In some embodiments, the T cellfunction is any one or more of increased IFNγ production from T cells,enhanced T cell survival, increased T cell proliferation, or rescue froman exhausted T cell phenotype. In some embodiments, the enhanced T cellfunction is greater upon administration of the multispecificantigen-binding construct, as compared to an agent that binds eitherPD-1 or a PD-1 ligand, or a cocktail comprising an agent that binds PD-1and an agent that binds a PD-1 ligand. In some embodiments, themultispecific antigen-binding construct is administered subcutaneously,intravenously, intradermally, intraperitoneally, orally, intramuscularlyor intracranially. In some embodiments, the multispecificantigen-binding construct binds to PD-1 and to PD-L1 expressed on thesurface of the same cell in the subject. In some embodiments, themultispecific antigen-binding construct binds to PD-1 expressed on thesurface of a first cell in the subject, and wherein the multispecificantigen-binding construct binds to PD-L1 expressed on the surface of asecond cell in the subject.

In some embodiments, the disclosure provides for an anti-PD1 antibody orantigen-binding fragment thereof, wherein the anti-PD-1 antibody orantigen-binding fragment comprises: (a) a heavy chain variable regioncomprising (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX₁X₂YAX₃X₄, whereinX₁=S, R, G, or N; X₂=D, S, N, A, R, or G; X₃=M or L; X₄=S, L, or N);(ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a CDRH3comprising SEQ ID NO: 72 (ARGLDFIVGX₅TGNDY, wherein X₅=A, Y, or R); and(b) a light chain variable region comprising: (i) a CDRL1 comprising SEQID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS);and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT). In someembodiments, the anti-PD-1 antibody or antigen-binding fragmentcomprises: (a) a heavy chain variable region comprising (i) a CDRH1comprising the amino acid sequence of any one of SEQ ID NOs: 73, 76, 77,78, 79, 80, 81, 82, 83, 84, or 86; (ii) a CDRH2 comprising the aminoacid sequence of SEQ ID NO: 71; and (iii) a CDRH3 comprising the aminoacid sequence of any one of SEQ ID NOs: 74, 75, or 85; and (b) a lightchain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9(RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii)a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT). In some embodiments, theheavy chain variable region comprises an amino acid sequence that is atleast 85% identical to the amino acid sequence of any one of SEQ ID NOs:87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99. In someembodiments, the light chain variable region comprises an amino acidsequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 59.

In some embodiments, the disclosure provides for an anti-PD-L1 antibodyor antigen-binding fragment thereof, wherein the anti-PD-L1 antibody orantigen-binding fragment comprises: a. a heavy chain variable regioncomprising (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2comprising SEQ ID NO: 2 (GGIIPX₁X₂GX₃ATYA, wherein X₁ is V or I; X₂ isF, L, or V; and X₃ is T or A); and (iii) a CDRH3 comprising SEQ ID NO: 3(ARLKX₁ELKDAFDI, wherein X₁ is G, F, or N); and b. a light chainvariable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4(RASQX₁ISSYLN, wherein X₁ is S, W, or Q); (ii) a CDRL2 comprising SEQ IDNO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 6 (X₁QSYSTPLT,wherein X₁ is Q or F). In some embodiments, the anti-PD-L1 antibody orantigen-binding fragment comprises: (a) a heavy chain variable regioncomprising (i) a CDRH1 comprising the amino acid sequence of any one ofSEQ ID NOs: 1, 14, 23, 36, or 122; (ii) a CDRH2 comprising the aminoacid sequence of any one of SEQ ID NOs: 11, 15, 16, 21, 24, 26, 27, 29,31, 33, or 34; and (iii) a CDRH3 comprising the amino acid sequence ofany one of SEQ ID NOs: 8, 17, 18, 19, 20, 22, 25, 28, 30, 32, or 37; and(b) a light chain variable region comprising: (i) a CDRL1 comprising theamino acid sequence of any one of SEQ ID NOs: 9, 12, or 13; (ii) a CDRL2comprising the amino acid sequence of SEQ ID NO: 5; and (iii) a CDRL3comprising the amino acid sequence of any one of SEQ ID NOs: 10, 38 or39. In some embodiments, the heavy chain variable region comprises anamino acid sequence that is at least 85% identical to the amino acidsequence of any one of SEQ ID NOs: 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 35. In some embodiments, thelight chain variable region comprises an amino acid sequence that is atleast 85% identical to the amino acid sequence of any one of SEQ ID NOs:59, 60, 61, 62, or 63.

In some embodiments, the disclosure provides for a method for treating aproliferative disorder in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of anyof the antibodies or antigen-binding constructs disclosed herein,thereby treating the proliferative disorder in the subject. In someembodiments, the proliferative disorder is cancer. In some embodiments,the cancer is selected from the group consisting of a hematologicalcancer, a neurological cancer, melanoma, breast cancer, lung cancer,head and neck cancer, a gastrointestinal cancer, liver cancer,pancreatic cancer, a genitourinary cancer, a bone cancer, and a vascularcancer.

In some embodiments, the disclosure provides for a method of enhancingan immune response in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of anyof the antibodies or antigen-binding constructs disclosed herein,thereby enhancing the immune response in the subject.

In some embodiments, any of the methods described herein can involvedetecting the presence or absence of PD-1 expression by one or morecells (or a population of cells, such as TILs) before and/or aftercontact with a multispecific antigen-binding molecule described herein.For example, any of the methods described herein can involve detectingthe presence or absence of PD-1 expression by one or more cells (or apopulation of cells, such as TILs) before and/or after administration ofa multispecific antigen-binding molecule described herein to a subject(e.g., a cancer patient). Such methods are useful, e.g., determining atherapeutically effective amount of the molecule for use in treatment ofa given patient or patient population. Methods of detecting thepresence, reduction in expression, and/or absence of PD-1 expression areknown to those of skill in the art, for example, using flow cytometry,Western blotting, ELISA, etc.

In another aspect, the disclosure features a method comprising measuringthe level of PD-1 expression by one or more cells (or a population ofcells, such as TILs) before and/or after contact with a multispecificantigen-binding molecule described herein. In some embodiments, themethod comprises measuring the level of PD-1 expression by one or morecells (or a population of cells, such as TILs) before and/or afteradministration of a multispecific antigen-binding molecule describedherein to a subject (e.g., a cancer patient).

In yet another aspect, the disclosure features a method comprisingmeasuring the level of PD-1 expression by one or more cells (or apopulation of cells, such as TILs) before and/or after contact with amultispecific antigen-binding molecule described herein. For example,any of the methods described herein can involve measuring the level ofPD-1 expression by one or more cells (or a population of cells, such asTILs) before and/or after administration of a multispecificantigen-binding molecule described herein to a subject (e.g., a cancerpatient). Such methods are useful for, among other things, detecting ormeasuring a biological effect of a molecule described herein on thesubject. In some embodiments, a reduction in the level of PD-1expression by an immune cell (e.g., TILs isolated from a patient)following treatment with a multispecific antigen-binding moleculedescribed herein indicates that the molecule has had a biological effectin the subject. In some embodiments, a reduction in the level of PD-1expression by an immune cell (e.g., TILs isolated from a patient)following treatment with a multispecific antigen-binding moleculedescribed herein indicates that the patient should receive one or moredoses of the molecule, or otherwise continue on a therapy comprising themolecule.

In yet another aspect, the disclosure features methods for determiningwhether a biological effect has occurred in a patient or population ofpatients treated with a multispecific antigen-binding molecule describedherein. The method comprises detecting the presence or amount of PD-1expression by one or more test immune cells (e.g., effector immunecells, such as those in the tumor microenvironment) obtained from apatient or patients who have been administered a multispecificantigen-binding molecule described herein, wherein a reduced level ofPD-1 expression (e.g., cell surface expression) of PD-1 by the one ormore immune cells relative to a control expression level (e.g., theexpression level of PD-1 by immune cells of the same histological typeas the test immune cells prior to administration of the molecule)indicates that a biological effect has occurred in the patient orpopulation of patients. In some embodiments, the method comprisesadministering the multispecific antigen-binding molecule prior to thedetecting. In some embodiments, the method comprises administering amultispecific antigen-binding molecule to the patient or population ofpatients in whom the occurrence of a biological effect has beendetermined. In some embodiments, control PD-1 expression level isapproximately the median or average expression level of PD-1 by immunecells of the same histological type in a population of subjects who havenot been diagnosed as having a cancer. In some embodiments, control PD-1expression level is approximately the median or average expression levelof PD-1 by immune cells of the same histological type in a population ofsubjects who have not been administered a multispecific antigen-bindingmolecule described herein and/or an agent that binds to and/or inhibitsPD-1.

In yet another aspect, the disclosure features a method for reducing theexpression of PD-1 by one or more immune cells in a subject (e.g., acancer patient), the method comprising administering a multispecificantigen-binding molecule described herein to a subject to thereby reducethe expression of PD-1 by one or more immune cells in the subject. Insome embodiments, the method comprises determining whether a reductionin PD-1 expression by one or more immune cells in the patient hasoccurred. In some embodiments, the method comprises obtaining from thesubject a biological sample (e.g., a tumor biopsy) comprising one ormore immune cells (e.g., after administration of the molecule to thesubject). In some embodiments, the method comprises measuring the levelof PD-1 expression by one or more immune cells in the biological sample.

In yet another aspect, the disclosure features a method for inhibitingbinding between PD-L1 and CD80 in a subject (e.g., a cancer patient),the method comprising administering a multispecific antigen-bindingmolecule described herein to a subject to thereby inhibit bindingbetween PD-L1 and CD80 in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the induction of interferon-gamma (IFNγ) in a mixedlymphocyte reaction (MLR) assaytreated with various antibody cocktailsor bispecific antibodies including a PembrolizumabxAtezolizumabbispecific, a NivolumabxAtezolizumab bispecific, a cocktail of KEYTRUDAand Atezolizumab, and a cocktail of Nivolumab and Atezolizumab, ascompared to KEYTRUDA alone. The results show the concentration of IFNγas pg/mL at the final concentrations of antibodies tested, as indicated.

FIG. 2 shows the induction of interferon-gamma (IFNγ) in mixedlymphocyte reaction (MLR) assaytreated with various monoclonal andbispecific antibodies including a PembrolizumabxNivolumab bispecific,AtezolizumabxAtezolizumab tetravalent fusion, Nivolumab, andAtezolizumab, as compared to KEYTRUDA alone. The results show theconcentration of IFNγ as pg/mL at the final concentrations of antibodiestested, as indicated.

FIG. 3 shows a schematic and amino acid sequence for aPembrolizumabxAtezolizumab bispecific. Separate sequences are given forthe Pembrolizumab_aglyco-IgG1-(G4S)₄ heavy chain (H chain; SEQ ID NO:104) and light chain (L chain; SEQ ID NO: 105) and for theAtezolizumab_FabH-(G4S)₄ heavy chain (H chain; SEQ ID NO: 106) and lightchain (L chain; SEQ ID NO: 107).

FIG. 4 shows a schematic and amino acid sequence for aNivolumabxAtezolizumab bispecific. Separate sequences are given for theNivolumab aglyco-IgG1-(G4S)₄ heavy chain (H chain; SEQ ID NO: 108) andlight chain (L chain; SEQ ID NO: 109) and for the AtezolizumabFabH-(G4S)₄ heavy chain (H chain; SEQ ID NO: 106) and light chain (Lchain; SEQ ID NO: 107).

FIG. 5 shows a schematic and amino acid sequence for a 949aglyco-IgG1×Atezolizumab bispecific. Separate sequences are given forthe 949_aglyco-IgG1-(G4S)₄ heavy chain (H chain; SEQ ID NO: 110) andlight chain (L chain; SEQ ID NO: 111) and for theAtezolizumab_FabH-(G4S)₄ heavy chain (H chain; SEQ ID NO: 106) and lightchain (L chain; SEQ ID NO: 107).

FIG. 6 shows a schematic and amino acid sequence for an AtezolizumabxNivolumab bispecific. Separate sequences are given for theAtezolizumab_aglyco-IgG1-(G4S)₄ heavy chain (H chain; SEQ ID NO: 112)and light chain (L chain; SEQ ID NO: 107) and Nivolumab_HC Fab-(G4S)₄heavy chain (H chain; SEQ ID NO: 113) and light chain (L chain; SEQ IDNO: 109).

FIG. 7 shows IFN-γ release, in pg/mL, in a mixed lymphocyte reaction(MLR) assay as a function of antibodies tested at various concentrations(right panel). These results indicate that bispecific antibodiesPD-1×PD-L1 (Pembrolizumabx Atezolizumab) or (NivolumabxAtezolizumab) inmultispecific format induce a greater IFN-γ response at femtomolarconcentrations, as compared to a cocktail of Pembrolizumab andAtezolizumab or Nivolumab and Atezolizumab. Size exclusionchromatography of the bispecific formats against mAb precursors is shown(left panel).

FIG. 8 shows an example of a workflow for identifying multispecific(e.g., bispecific) antibodies that demonstrate synergy. The processincludes an unbiased screen of checkpoint blocker combinations in amixed lymphocyte reaction (MLR) assay, which measures IFN-γ release, inpg/mL, at various concentrations. In the second step of the illustratedworkflow, common light chain bispecifics were generated to further testtheir efficacy; here, various bispecific formats are depicted. Theidentified bispecific formats outperform known PD-1 blockers in a T-cellactivation assay.

FIGS. 9A-9B show that Bispecific 3 induced higher killing ofK562-CD32-PDL1 target cells (FIG. 9A) and increased IFNγ production(FIG. 9B) by CD3/CD28 expanded T-cells in an antigen non-specific T cellassay, as compared to both an isotype control antibody and KEYTRUDA.This increased killing by Bispecific 3 was seen even at lowconcentrations of 0.01 nM.

FIGS. 10A-10B show an effect of Bispecific 3 on tumor cell killing. FIG.10A shows that Bispecific 3 increased the specific killing ofK562-A2-CMV-PDL1 tumor antigen target cells by CMV specific T-cells atlow concentrations of 0.001 through 0.01 nM, as compared to bothKEYTRUDA and the combination of mAb1 and mAb28, indicating thatBispecific 3 can be used to mediate antigen-specific killing of targetcells at lower doses. FIG. 10B shows that Bispecific 3 was moreeffective than either KEYTRUDA or the combination of mAb1 and mAb28 inthe specific killing of Raji-A2-CMV-PDL1 cell tumor antigen target cellsby CMV specific T-cells at low concentrations of 0.001 nM, againindicating that Bispecific 3 can be used to mediate antigen-specifickilling of target cells at lower doses.

FIG. 11 shows that Bispecific 3 induced more IL-2 than KEYTRUDA at alltested doses in an SEA stimulation assay. Importantly, Bispecific 3induced increased IL-2 production starting at lower concentrations ofantibody as compared to both KEYTRUDA and mAb1 and mAb28.

FIGS. 12A-12G demonstrate that Bispecific 3 has a unique ability tocause the internalization and subsequent degradation or loss ofexpression of PD-1, and that this property is dependent on engagement ofboth the PD-1 and PD-L1 targeting arms of the molecule. FIGS. 12A-12Bdemonstrate that only Bispecific 3 results in internalization andsubsequent degradation or loss of expression of PD-1, when compared toisotype control, KEYTRUDA, mAb1 and mAb28, Atezolizumab, or Atezolizumaband KEYTRUDA. Additionally, as shown in FIG. 12C, when the anti-PD-L1antibody, mAb1, was added at 50 nM to wells 5 minutes before addingBispecific 3, the ability of Bispecific 3 to drive PD-1 internalizationwas lost. This suggests that both arms of Bispecific 3 should be engagedto drive PD-1 loss of expression and/or internalization and/ordegradation. FIG. 12D shows that treatment with Bispecific 3 increasesthe amount of PD-1 in the supernatant when both binding arms of thebispecific are engaged concurrently. This effect is lost when the PD-L1targeting arm is blocked by mAb1. This suggests that Bispecific 3increases shedding of PD-1 into the supernatant. FIG. 12E demonstratesthat the valency of the binding arms influences the degree of the lossof PD-1 expression. Bispecific 5 was made having a first N-terminal Fabbinding PD-L1 based on the VH and VL sequences of mAb1 and having asecond N-terminal Fab binding PD-1 based on the VH and VL sequences ofmAb28. In other words, Bispecific 5 has one monovalent arm binding PD-L1and one monovalent arm binding PD-1, as compared to Bispecific 3, whichhas bivalent arms binding PD-L1 and bivalent arms binding PD-1. Asshown, loss of PD-1 expression starts to occur at higher doses ofBispecific 5 (bivalent) versus Bispecific 3 (tetravalent), suggestingthat the increased valency of Bispecific 3 is responsible for thisdifference. FIG. 12F demonstrates that pretreatment with Batimastat, abroad-spectrum inhibitor of multiple MMPs and ADAMs, sheddases orproteases responsible for cleaving proteins off the plasma membrane ofcells, greatly reduces the amount of cell-associated PD-1 loss,suggesting that PD-1 loss or shedding is due to cleavage by an MMP orADAM protease. FIG. 12G suggests that Bispecific 3 drives loss ofcell-surface PD-1 expression primarily when it binds to PD-1 and PD-L1that are in the trans configuration, i.e., are being expressed bydifferent cells.

FIGS. 13A-13B shows in vivo results using Bispecific 3. FIG. 13A depictsa schematic of the experimental protocol. FIG. 13B demonstrates thatboth Bispecific 3 and the combination mAb1 and mAb28 groups hadsignificant delays in tumor growth as compared to both the isotype andKEYTRUDA groups. In addition, at day 24, there was a significantdivergence between the Bispecific 3 group and the group treated with acombination of mAb1 and mAb28, with Bispecific 3 causing a greater delayin tumor growth as compared to the combination. The No T cell transfergroup had tumors that grew more aggressively than any group containing Tcells. In this model, KEYTRUDA gave no benefit in delaying KACP tumorgrowth as compared to the isotype control.

FIGS. 14A-14B shows in vivo results using Bispecific 3 in aK562-A2-CMV-PD-L1 tumor mouse model. FIGS. 14A and 14B are graphsillustrating that, while each of the different treatment groups resultedin a delay in average tumor growth as compared to untreated mice,treatment with T cells and Bispecific 3 resulted in the greatest delayin average tumor growth over time. The No T cell transfer group hadtumors that grew more aggressively than any group containing T cells. Inthis model, KEYTRUDA gave no benefit in delaying K562-A2-CMV-PD-L1 tumorgrowth as compared to the isotype control.

FIGS. 15A-15B illustrate in vivo results using Bispecific 3 in severalsyngeneic tumor models. FIG. 15A is a graph showing that treatment of anEMT-6 syngeneic tumor model with Bispecific 3 resulted in a greaterdelay in tumor growth as compared to the control treatment. FIG. 15B isa graph showing that treatment of an MB49 syngeneic tumor model withBispecific 3 resulted in a greater delay in tumor growth as compared tothe control treatment.

FIGS. 16A-16B show in vivo results using Bispecific 1 in an MC38-hPD-L1model in humanized PD-1/PD-L1 transgenic mice. FIG. 16A shows that bothKEYTRUDA and Bispecific 1 treatment effectively controlled tumor growthin the MC38-hPD-L1 tumor mice as compared to control-treated mice. FIG.16B is a survival graph and illustrates that Bispecific 1 increased thesurvival of MC38-PD-L1 tumor mice as compared to control-treated mice.

FIGS. 17A-17F illustrate in vivo results using Bispecific 3 in aB16F10-hPD-L1 model. FIG. 17A is a series of graphs each showing theeffect of different treatments on tumor growth measured at a 15-daycutoff in B16F10-HuPD-L1 mice. Different groups of mice (n=8) weretreated with Bispecific 3, KEYTRUDA, Avelumab, KEYTRUDA+Avelumabcombination, or isotype control antibodies. FIG. 17A shows individualtumor volume traces for each group. Metastases were identified inmultiple mice that died prior to the tumor-sizing cutoff. FIG. 17B showsdifferences in mean tumor volume across treatment groups, demonstratingthat by 15 days after tumor cell inoculation, Bispecific 3 treatmentdelayed average tumor growth significantly longer than any of the othertreatments tested in B16F10-HuPD-L1 mice. ****, P<0.0001; **, P<0.01, *,P<0.05, Two-way ANOVA and Tukey's multiple comparisons test. FIG. 17B isa graph comparing the effect of different treatments on tumor volumemeasured at a 15-day cutoff in B16F10-HuPD-L1 mice. Metastases wereidentified in multiple mice that died prior to the tumor-sizing cutoff.As shown in FIG. 17B, Bispecific 3 treatment delayed average tumorgrowth significantly longer than any of the other treatments tested inB16F10-hPD-L1 mice. FIG. 17C is a survival graph and illustrates thattreatment with Bispecific 3 increased the survival of B16F10-hPD-L1tumor mice as compared to survival with any of the other treatmentstested. FIG. 17D shows that by 21 days after tumor cell inoculation,Bispecific 3 treatment continued to delay average tumor growthsignificantly better than treatment with KEYTRUDA in B16F10-hPD-L1 mice.FIG. 17E is a survival graph and illustrates that treatment withBispecific 3 increased the survival of B16F10-HuPD-L1 tumor mice ascompared to survival with any of the other treatments tested. FIG. 17Fprovides a Table showing the number of tumor-free mice for each of thedifferent treatment groups. The group treated with Bispecific 3 had 3mice that were tumor-free, while the combination of KEYTRUDA andavelumab had 1 mouse that was tumor-free.

FIGS. 18A-18D demonstrate that Bispecific 3 has drug-like properties(DLP's) similar to a well-behaved monoclonal antibody and maintainsparental PD-1 and PD-L1 binding. FIG. 18A shows that Bispecific 3 showssimilar binding to CHO cells expressing human PD-1 as parental clonemAb28 (top) and to CHO cells expressing human PD-L1 as parental clonemAb1 (bottom). FIG. 18B shows that Bispecific 3 shows similar binding toCHO cells expressing cynomolgus PD-1 as parental clone mAb28 (top) andto CHO cells expressing cynomolgus PD-L 1 as parental clone mAb1(bottom). FIG. 18C shows that Bispecific 3 shows similar binding to CHOcells expressing mouse PD-1 as parental clone mAb28 (top) and to CHOcells expressing mouse PD-L1 as parental clone mAb1 (bottom). FIG. 18Dshows a size-exclusion chromatography trace of Bispecific 3 afterProtein A chromatography (top) demonstrating a single peak with greaterthan 98% purity and a differential scanning fluorimetry (DSF) trace ofBispecific 3 (bottom) demonstrating that the molecule has high thermalstability.

DETAILED DESCRIPTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which thisdisclosure pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodologies by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the singular forms “a,” “an,” and “the” include theplural referents unless the context clearly indicates otherwise.

In the specification and claims, the term “about” is used to modify, forexample, the quantity of an ingredient in a composition, concentration,volume, process temperature, process time, yield, flow rate, pressure,and like values, and ranges thereof, employed in describing theembodiments of the disclosure. The term “about” refers to variation inthe numerical quantity that can occur, for example, through typicalmeasuring and handling procedures used for making compounds,compositions, concentrates or use formulations; through inadvertenterror in these procedures; through differences in the manufacture,source, or purity of starting materials or ingredients used to carry outthe methods, and like proximate considerations. The term “about” alsoencompasses amounts that differ due to aging of a formulation with aparticular initial concentration or mixture, and amounts that differ dueto mixing or processing a formulation with a particular initialconcentration or mixture. Where modified by the term “about” the claimsappended hereto include equivalents to these quantities. If there areuses of the term which are not clear to persons of ordinary skill giventhe context in which it is used, “about” will mean up to plus or minus10% of the particular value.

With regard to the binding of an antigen-binding protein/region/arm to atarget molecule, the terms “specific binding,” “specifically binds to,”“specific for,” “selectively binds,” and “selective for” a particularantigen (e.g., a polypeptide target) or an epitope on a particularantigen mean binding that is measurably different from a non-specific ornon-selective interaction. Specific binding can be measured, forexample, by determining binding of a molecule compared to binding of acontrol molecule. Specific binding can also be determined by competitionwith a control molecule that is similar to the target, such as an excessof non-labeled target. In that case, specific binding is indicated ifthe binding of the labeled target to a probe is competitively inhibitedby the excess non-labeled target.

The term “epitope” means a component of an antigen capable of specificbinding to an antigen-binding protein. Epitopes frequently consist ofsurface-accessible amino acid residues and/or sugar side chains and canhave specific three-dimensional structural characteristics, as well asspecific charge characteristics. Conformational and non-conformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents. An epitope cancomprise amino acid residues that are directly involved in the binding,and other amino acid residues, which are not directly involved in thebinding. The epitope to which an antigen-binding protein binds can bedetermined using known techniques for epitope determination such as, forexample, testing for antigen-binding protein binding to antigen variantswith different point-mutations.

Percent “identity” between a polypeptide sequence and a referencesequence, is defined as the percentage of amino acid residues in thepolypeptide sequence that are identical to the amino acid residues inthe reference sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGAsoftware. In some embodiments, alignment is performed using the CLUSTALOMEGA software. Those skilled in the art can determine appropriateparameters for aligning sequences, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared.

The term “capable of” as used herein means that an agent or method(e.g., any of the multispecific antigen-binding constructs or methodsdisclosed herein) has the ability to achieve a specified property in theappropriate context (as would be understood by the skilled worker), butis not required to be associated with that property at any particularmoment in time. For example, any of the multispecific antigen-bindingconstructs disclosed herein may be capable of binding PD-1 and/or PD-L1when administered to cells expressing PD-1 and/or PD-L1, but theconstructs would not be expected to bind PD-1 and/or PD-L1 when theconstructs are in a composition devoid of PD-1 or PD-L1 protein.

A “conservative substitution” or a “conservative amino acidsubstitution,” refers to the substitution of one or more amino acidswith one or more chemically or functionally similar amino acids.Conservative substitution tables providing similar amino acids are wellknown in the art. Polypeptide sequences having such substitutions areknown as “conservatively modified variants,” or “variants.” Suchconservatively modified variants are in addition to and do not excludepolymorphic variants, interspecies homologs, and alleles. Some examplesof conservative substitutions can be found, for example, in Creighton,Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H.Freeman & Co., New York, N.Y.

A polypeptide disclosed herein can comprise an amino acid sequence whichis not naturally occurring. Such variants necessarily have less than100% sequence identity or similarity with the starting molecule. Incertain embodiments, the variant will have an amino acid sequence fromabout 75% to less than 100% amino acid sequence identity or similaritywith the amino acid sequence of the starting (e.g., naturally-occurringor wild-type) polypeptide, more preferably from about 80% to less than100%, more preferably from about 85% to less than 100%, more preferablyfrom about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%) and most preferably from about 95% to less than 100%,e.g., over the length of the variant molecule.

An “antibody,” as used herein, can refer to an intact antibody (e.g., anintact immunoglobulin) and antibody portion, for example, anantigen-binding portion. Antigen-binding portions comprise at least oneantigen-binding domain. One example of an antigen-binding domain is anantigen-binding domain formed by a VH-VL dimer. Antibodies andantigen-binding portions can be described by the antigen to which theyspecifically bind. For example, a PD-L1 antibody, or anti-PD-L1antibody, is an antibody that specifically binds to PD-L1.

The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability (hypervariable regions (HVRs), also calledcomplementarity determining regions (CDRs)) interspersed with regionsthat are more conserved. The more conserved regions are called frameworkregions (FRs). Each V_(H) and V_(L) generally comprises three CDRs andfour FRs, arranged in the following order (from N-terminus toC-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved inantigen-binding, and confer antigen specificity and binding affinity tothe antibody. (See Kabat et al. (1991) Sequences of Proteins ofImmunological Interest 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md.)

CDRs are involved in antigen binding and confer antigen specificity andbinding affinity to the antibody. There are three CDRs in each of thevariable domains of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the variable domains. Theterm “CDR set” as used herein refers to a group of three CDRs that occurin a single heavy or light chain variable domain capable of binding atarget antigen. The exact boundaries of these CDRs have been defineddifferently according to different systems. The three heavy chain CDRscan be referred to as CDRH1, CDRH2, and CDRH3, and the three light chainCDRs can be referred to as CDRL1, CDRL2, and CDRL3.

The system described by Kabat, also referred to as “numbered accordingto Kabat,” “Kabat numbering,” “Kabat definitions,” and “Kabat labeling,”provides an unambiguous residue numbering system applicable to anyvariable domain of an antibody, and provides precise residue boundariesdefining the three CDRs of each chain. (Kabat et al., Sequences ofProteins of Immunological Interest, National Institutes of Health,Bethesda, Md. (1987) and (1991), the contents of which are incorporatedby reference in their entirety. These CDRs are referred to as Kabat CDRsand comprise about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3)in the light chain variable domain, and 31-35 (CDR1), 50-65 (CDR2) and95-102 (CDR3) in the heavy chain variable domain. When the CDRs aredefined according to Kabat, the light chain FR residues are positionedat about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107(LCFR4) and the heavy chain FR residues are positioned about at residues1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in theheavy chain residues. The “EU index as in Kabat” refers to the residuenumbering of the human IgG1 EU antibody.

Other CDR numbering systems are also used in the art (see, for example,Table A). Chothia and coworkers found that certain sub-portions withinKabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.(Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothia et al.(1989) Nature 342: 877-883). These sub-portions were designated as L1,L2, and L3 or H1, H2, and H3 where the “L” and the “H” designates thelight chain and the heavy chains regions, respectively. These CDRs canbe referred to as “Chothia CDRs,” “Chothia numbering,” or “numberedaccording to Chothia,” and comprise about residues 24-34 (CDR1), 50-56(CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26-32(CDR1), 52-56 (CDR2) and 95-102 (CDR3) in the heavy chain variabledomain. Mol. Biol. 196:901-917 (1987).

The system described by MacCallum, also referred to as “numberedaccording to MacCallum,” or “MacCallum numbering” comprises aboutresidues 30-36 (CDR1), 46-55 (CDR2) and 89-96 (CDR3) in the light chainvariable domain, and 30-35 (CDR1), 47-58 (CDR2) and 93-101 (CDR3) in theheavy chain variable domain. MacCallum et al. ((1996) J. Mol. Biol.262(5):732-745).

The system described by AbM, also referred to as “numbering according toAbM,” or “AbM numbering” comprises about residues 24-34 (CDR1), 50-56(CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26-35(CDR1), 50-58 (CDR2) and 95-102 (CDR3) in the heavy chain variabledomain.

The IMGT (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) numbering ofvariable regions can also be used, which is the numbering of theresidues in an immunoglobulin variable heavy or light chain according tothe methods of the IIMGT, as described in Lefranc, M.-P., “The IMGTunique numbering for immunoglobulins, T cell Receptors and Ig-likedomains”, The Immunologist, 7, 132-136 (1999), and is expresslyincorporated herein in its entirety by reference. As used herein, “IMGTsequence numbering” or “numbered according to IMTG,” refers to numberingof the sequence encoding a variable region according to the IMGT. Forthe heavy chain variable domain, when numbered according to IMGT, thehypervariable region ranges from amino acid positions 27 to 38 for CDR1,amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to117 for CDR3. For the light chain variable domain, when numberedaccording to IMGT, the hypervariable region ranges from amino acidpositions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, andamino acid positions 105 to 117 for CDR3.

In some embodiments of the constructs and antigen-binding arms describedherein, the CDRs recited herein comprise about residues 24-34 (CDR1),49-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and27-35 (CDR1), 49-60 (CDR2) and 93-102 (CDR3) in the heavy chain variabledomain, when numbered according to Chothia numbering. In someembodiments, CDR2 in the light chain variable domain can comprise aminoacids 49-56, when numbered according to Chothia numbering.

TABLE A CDR Definitions CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 Kabat 31-3550-65  95-102 24-34 50-56 89-97 Alternative 27-35 49-60  93-102 24-3450-56 89-97 CDRs numbered according to Chothia Chothia 26-32 52-56 95-102 24-34 50-56 89-97 or 50-56 MacCallum 30-35 47-58  93-101 30-3646-55 89-96 AbM 26-35 50-58  95-102 24-34 50-56 89-97 IMGT 27-38 56-65105-117 27-38 56-65 105-117

Preferred methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the presently disclosed methodsand compositions. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety.

Various aspects of the invention are described in further detail below.Additional definitions are set out throughout the specification.

The immune system has the capability of recognizing and eliminatingtumor cells; however, tumors can use multiple strategies to evadeimmunity. Recent studies have shown that inhibitory immune checkpointmolecules promote cancer progression through various antitumorinhibitory mechanisms. Blockade of immune checkpoints is one of theapproaches to activating or reactivating therapeutic antitumor immunity.Various ligands have been described for a number of cognate inhibitoryimmune checkpoint receptors. Reviewed in, e.g., Nair & Elkord,Immunology & Cell Biology (2018), 96:21-33; and Jenkins et al., BritishJ. of Cancer (2017), 118:9-16.

The Programmed Death 1 (PD-1) protein is an inhibitory member of theextended CD28/CTLA-4 family of T cell regulators (Okazaki et al. (2002)Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) J. Immunol.170:711-8). Other members of the CD28 family include CD28, CTLA-4, ICOSand BTLA. PD-1 is suggested to exist as a monomer, lacking the unpairedcysteine residue characteristic of other CD28 family members. PD-1 isexpressed on activated B cells, T cells, and monocytes.

The PD-1 gene encodes a 55 kDa type I transmembrane protein (Agata etal. (1996) Int Immunol. 8:765-72). Although structurally similar toCTLA-4, PD-1 lacks the MYPPY motif that is important for B7-1 and B7-2binding. Two ligands for PD-1 have been identified, PD-L1 (B7-H1) andPD-L2 (B7-DC), that have been shown to downregulate T cell activationupon binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34;Carter et al. (2002) Eur. J. Immunol. 32:634-43). Both PD-L1 and PD-L2are B7 homologs that bind to PD-1, but do not bind to other CD28 familymembers. PD-L1 is abundant in a variety of human cancers (Dong et al.(2002) Nat. Med. 8:787-9).

PD-1 is known as an immunoinhibitory protein that negatively regulatesTCR signals (Ishida, Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. etal. (Epub 2006 Dec. 29) Immunol. Immunother. 56(5):739-745). Theinteraction between PD-1 and PD-L1 can act as an immune checkpoint,which can lead to, e.g., a decrease in tumor infiltrating lymphocytes, adecrease in T-cell receptor mediated proliferation, and/or immuneevasion by cancerous cells (Dong et al. (2003) J. Mol. Med. 81:281-7;Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi etal. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression can bereversed by inhibiting the local interaction of PD-1 with PD-L1 orPD-L2; the effect is additive when the interaction of PD-1 with PD-L2 isblocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

PD-L1, also known as cluster of differentiation 274 (CD274) or B7homolog 1 (B7-H1) is a 40 kDa type 1 transmembrane protein that plays arole in suppressing the immune system during particular events such aspregnancy, tissue allografts, autoimmune disease and other diseasestates such as hepatitis. By way of example, human PD-L1 comprises theamino acid sequence of SEQ ID NO: 115 (UniProt Q9NZQ7). Normally theimmune system reacts to foreign antigens that are associated withexogenous or endogenous danger signals, which triggers a proliferationof antigen-specific CD8+ T cells and/or CD4+ helper cells. In cancer,PD-L1 expressed on cancer cells binds to its ligand PD-1 on immuneeffector cells, for example, T cells. The binding of PD-L1 to PD-1transmits an inhibitory signal that reduces the proliferation ofantigen-specific T-cells in lymph nodes, while simultaneously reducingapoptosis in regulatory T cells (anti-inflammatory, suppressive Tcells). The PD-1/PD-L1 interaction also induces apoptosis oftumor-specific T cells, promotes the differentiation of CD4⁺ T cellsinto Foxp3⁺ regulatory T cells, and promotes resistance of tumor cellsto cytotoxic T lymphocyte (CTL) attack, thus allowing tumors to evadethe host immune system.

The present disclosure relates to compositions and methods forinhibiting tumor evasion by reducing immune checkpoint suppression thatresults from the interaction between PD-1 and its ligand (e.g., PD-L1and/or PD-L2). In particular, provided herein are compositionscomprising novel multispecific and multivalent constructs, such as abispecific and tetravalent construct, that block the interaction betweenPD-1 and its ligand (e.g., PD-1 and/or PD-L2) while promoting theinteraction of (bridging) the cells on which PD-1 and its ligand areexpressed. Such compositions of the present disclosure with the capacityto “block and bridge” have increased potency in vitro and in vivo andstrongly enhance, for example, T cell proliferation, IFNγ productionand/or secretion, the cytolytic activity of T cells, and/or rescue Tcells from functional exhaustion, to provide superior anti-tumorefficacy (a biological effect which can be manifested by various means,including, but not limited to, e.g., a decrease in tumor volume, adecrease in the number of tumor cells, a decrease in tumor cellproliferation, and/or a decrease in tumor cell survival), as compared tocombinations of individual antibodies, as well as compared to clinicalcheckpoint blockade agents. Also provided herein are novel monoclonalanti-PD-1 antibodies and antigen-binding fragments thereof, and novelmonoclonal anti-PD-L1 antibodies and antigen-binding fragments thereof,for use in such multispecific and multivalent constructs. Some of thesenovel monoclonal anti-PD-1 antibodies and novel monoclonal anti-PD-L1antibodies share a common light chain, thereby allowing for thegeneration of multispecific and multivalent constructs having excellentdrug-like properties and manufacturability, as well as affinitiessimilar to their parental antibodies.

Accordingly, the disclosure provides a multispecific antigen-bindingconstruct comprising at least two antigen-binding arms or units ofantigen-binding, wherein a first arm or unit of antigen-binding bindsPD-1 expressed by an immune cell, and a second arm or unit ofantigen-binding binds one or more PD-1 ligands (e.g., PD-L1 and/orPD-L2) which is expressed by a second cell. In some embodiments, themultispecific antigen-binding construct blocks the interaction of PD-1and its ligand. In some embodiments, the multispecific antigen-bindingconstruct bridges the cells on which PD-1 and its ligand are expressedto promote the interaction and/or efficacy of the immune cell thatexpresses PD-1. In some embodiments, at least one of the antigen-bindingarms is bivalent for PD-1. In some embodiments, at least one of theantigen-binding arms is bivalent for PD-L1. In some embodiments, atleast one of the antigen-binding arms is bivalent for PD-1, and at leastone of the antigen-binding arms is bivalent for PD-L1. In someembodiments, the multispecific antigen-binding construct comprises atleast two units of antigen-binding that bind PD-1. In some embodiments,the multispecific antigen-binding construct comprises two units ofantigen-binding that bind PD-1. In some embodiments, the multispecificantigen-binding construct comprises at least two units ofantigen-binding that bind a PD-1 ligand, such as PD-L1 or PD-L2. In someembodiments, the multispecific antigen-binding construct comprises twounits of antigen-binding that bind a PD-1 ligand, such as PD-L1 orPD-L2. In some embodiments, the multispecific antigen-binding constructcomprises at least four units of antigen-binding, wherein two units ofantigen-binding bind PD-1 and two units of antigen-binding bind a PD-1ligand, such as PD-L1 or PD-L2. In some embodiments, the multispecificantigen-binding construct comprises four units of antigen-binding,wherein two units of antigen-binding bind PD-1 and two units ofantigen-binding bind a PD-1 ligand, such as PD-L1 or PD-L2. In someembodiments of any of the aspects described herein, the construct is abispecific antibody. In some embodiments, the bispecific antibody is anantagonist of both PD-1 and PD-1 ligand. In some embodiments, theconstruct comprises a common light chain. In some embodiments, one orboth of the antigen-binding arms is an aptamer. In some embodiments, oneor both of the antigen-binding arms is a protein other than an antibody.In some embodiments, the construct comprises at least two antibodies. Insome embodiments, at least one of the antigen-binding arms is a bivalentantibody specific for PD-1. In some embodiments, at least one of theantigen-binding arms is a bivalent antibody specific for PD-L1. In someembodiments, at least one of the antigen-binding arms is a bivalentantibody specific for PD-1, and at least one of the antigen-binding armsis a bivalent antibody specific for PD-L1, such that the construct istetravalent. In some embodiments, the bispecific antibody binds twodifferent epitopes on PD-1. In some embodiments, the bispecific antibodybinds two different epitopes on the PD-1 ligand. Also provided herein,in some aspects, are novel isolated antibodies and antigen-bindingportions thereof that specifically bind PD-L1 or PD-1. In someembodiments, these novel isolated antibodies and antigen-bindingportions thereof, such as CDRs, variable heavy chains, and/or variablelight chains, that specifically bind PD-L1 or PD-1 can be used in one ormore arms or units of antigen-binding of the multispecificantigen-binding constructs described herein.

Accordingly, as described herein, the disclosed multispecificantigen-binding constructs include bispecific, tri specific,tetraspecific, or multispecific antibodies or antigen-binding portionsthereof. The described multispecific constructs are preferably bivalentfor at least one, preferably both, antigen-binding arm(s), i.e., arebispecific and trivalent, or bispecific and tetravalent molecules. Themultispecific constructs described herein can, in various aspects andembodiments, comprise one or more antibodies and/or antigen-bindingportions thereof. For example, an antigen-binding arm can comprise avariable heavy and/or variable light chain, or complementaritydetermining regions (CDRs) thereof, of a given antibody to PD-1 and/or agiven antibody to PD-L1. Accordingly, in some embodiments of any of theaspects described herein, the first antigen-binding arm, secondantigen-binding arm, first unit of antigen-binding, second unit ofbinding, or any combination thereof, can comprise an antibody or anantigen-binding portion thereof. In some embodiments of any of theaspects described herein, the first antigen-binding arm, secondantigen-binding arm, first unit of antigen-binding, second unit ofbinding, or any combination thereof, is an antibody or anantigen-binding portion thereof.

A. PD-L1 Antagonists

In some aspects and embodiments, the disclosure provides for anti-PD-L1antagonists. In some embodiments, the anti-PD-L1 antagonist is any ofthe anti-PD-L1 antibodies or antigen-binding molecules disclosed herein.In some embodiments, the anti-PD-L1 antibody or antigen-binding moleculeis not a part of a multispecific antigen-binding construct, i.e., theanti-PD-L1 antibody or antigen-binding molecule is not a part of aprotein construct that binds to multiple epitopes. In some embodiments,the anti-PD-L1 antibody or antigen-binding portion can be combined witha different antibody or antigen-binding portion to form a multispecificantigen-binding construct. In some embodiments, the multispecificantigen-binding construct is capable of binding an epitope on PD-L1 andan epitope on another protein. In some embodiments, the epitope on theother protein is on PD-1.

Accordingly, in some aspects, provided herein are antibodies orantigen-binding portions thereof that specifically bind PD-L1. In someaspects, the antibody or antigen-binding portion thereof thatspecifically binds PD-L1 comprises (a) a heavy chain variable regioncomprising (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2comprising SEQ ID NO: 2 (GGIIPX₁X₂GX₃ATYA, wherein X₁ is V or I; X₂ isF, L, or V; and X₃ is T or A); and (iii) a CDRH3 comprising SEQ ID NO: 3(ARLKX₁ELKDAFDI, wherein X₁ is G, F, or N); and (b) a light chainvariable region comprising: (i) a CDRL1 comprising SEQ ID NO: 4(RASQX₁ISSYLN, wherein X₁ is S, W, or Q); (ii) a CDRL2 comprising SEQ IDNO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO: 6 (X₁QSYSTPLT,wherein X₁ is Q or F).

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 7 (GGIIPILGAATYA) and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI).In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 7 (GGIIPILGAATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI);CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5(AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). Arepresentative antibody having such heavy and light chain variable CDRregions is mAb1.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 11 (GGIIPVFGTATYA) and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI).In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI);CDRL1 comprises SEQ ID NO: 12 (RASQWISSYLN); CDRL2 comprises SEQ ID NO:5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). Arepresentative antibody having such heavy and light chain variable CDRregions is mAb2. In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN),CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ IDNO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 13 (RASQQISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb3.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI);CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5(AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). Arepresentative antibody having such heavy and light chain variable CDRregions is mAb4. Another representative antibody having such heavy andlight chain variable CDR regions is mAb24.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 14 (GTFSSYAFS), CDRH2 comprises SEQID NO: 11 (GGIIPVFGTATYA) and CDRH3 comprises SEQ ID NO: 8(ARLKGELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 14 (GTFSSYAFS),CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ IDNO: 8 (ARLKGELKDAFDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10(QQSYSTPLT). A representative antibody having such heavy and light chainvariable CDR regions is mAb5.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 15 (GGIIPIFGIANYA) and CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI).In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 15 (GGIIPIFGIANYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI);CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5(AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). Arepresentative antibody having such heavy and light chain variable CDRregions is mAb6.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 16 (GGIIPNFGTATYA) and CDRH3 comprises SEQ ID NO: 17(ARLKGELKGAGDI). In some embodiments of these aspects and all suchaspects described herein SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQID NO: 16 (GGIIPNFGTATYA), CDRH3 comprises SEQ ID NO: 17(ARLKGELKGAGDI); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10(QQSYSTPLT). A representative antibody having such heavy and light chainvariable CDR regions is mAb7.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 18(ARLKFELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN),CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ IDNO: 18 (ARLKFELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb8.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 19(ARLKGELKDAFDE). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN),CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ IDNO: 19 (ARLKGELKDAFDE), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb9.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 20(ARLKNELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN),CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ IDNO: 20 (ARLKNELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb10.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 21 (GGVIPFLGTANYA), and CDRH3 comprises SEQ ID NO: 22(ARLKGILKDALDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN),CDRH2 comprises SEQ ID NO: 21 (GGVIPFLGTANYA), CDRH3 comprises SEQ IDNO: 22 (ARLKGILKDALDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb11.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQID NO: 24 (GGIIPIVGIANYA), and CDRH3 comprises SEQ ID NO: 8(ARLKGELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS),CDRH2 comprises SEQ ID NO: 24 (GGIIPIVGIANYA), CDRH3 comprises SEQ IDNO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10(QQSYSTPLT). A representative antibody having such heavy and light chainvariable CDR regions is mAb12.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 25(ARLKGEFKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS),CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ IDNO: 25 (ARLKGEFKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb13.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQID NO: 26 (GRIIPLFGTAHYA), and CDRH3 comprises SEQ ID NO: 8(ARLKGELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS),CDRH2 comprises SEQ ID NO: 26 (GRIIPLFGTAHYA), CDRH3 comprises SEQ IDNO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10(QQSYSTPLT). A representative antibody having such heavy and light chainvariable CDR regions is mAb14.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQID NO: 27 (GRINPILGTANYA), and CDRH3 comprises SEQ ID NO: 28(ARLKGELKDAFSI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS),CDRH2 comprises SEQ ID NO: 27 (GRINPILGTANYA), CDRH3 comprises SEQ IDNO: 28 (ARLKGELKDAFSI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb15.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 29 (GRIIPIFGTADYA), and CDRH3 comprises SEQ ID NO: 8(ARLKGELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN),CDRH2 comprises SEQ ID NO: 29 (GRIIPIFGTADYA), CDRH3 comprises SEQ IDNO: 8 (ARLKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO: 10(QQSYSTPLT). A representative antibody having such heavy and light chainvariable CDR regions is mAb16.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS), CDRH2 comprises SEQID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 30(ARLKGELKCAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 23 (GTFSSYAIS),CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ IDNO: 30 (ARLKGELKCAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT).

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 122 (GTKSSYAIS), CDRH2 comprises SEQID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 30(ARLKGELKCAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 122 (GTKSSYAIS),CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ IDNO: 30 (ARLKGELKCAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb17.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 31 (GGIIPILGTATYA), and CDRH3 comprises SEQ ID NO: 32(ARRKGELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN),CDRH2 comprises SEQ ID NO: 31 (GGIIPILGTATYA), CDRH3 comprises SEQ IDNO: 32 (ARRKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb18.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 33 (GGIIPIVATANYA), and CDRH3 comprises SEQ ID NO: 32(ARRKGELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN),CDRH2 comprises SEQ ID NO: 33 (GGIIPIVATANYA), CDRH3 comprises SEQ IDNO: 32 (ARRKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb19.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 34 (GGIIPIFGKATYA), and CDRH3 comprises SEQ ID NO: 32(ARRKGELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN),CDRH2 comprises SEQ ID NO: 34 (GGIIPIFGKATYA), CDRH3 comprises SEQ IDNO: 32 (ARRKGELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb20.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 36 (GPFRSHAVS), CDRH2 comprises SEQID NO: 11 (GGIIPVFGTATYA), and CDRH3 comprises SEQ ID NO: 37(ARLKSELKDAFDI). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 36 (GPFRSHAVS),CDRH2 comprises SEQ ID NO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ IDNO: 37 (ARLKSELKDAFDI), CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb21.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI);CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5(AASSLQS); and CDRL3 comprises SEQ ID NO: 38 (FQSYSTPLT). Arepresentative antibody having such heavy and light chain variable CDRregions is mAb22.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI);CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5(AASSLQS); and CDRL3 comprises SEQ ID NO: 39 (QQSYSTILT). Arepresentative antibody having such heavy and light chain variable CDRregions is mAb23.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 1 (GTFSSYAIN), CDRH2 comprises SEQ IDNO: 11 (GGIIPVFGTATYA), CDRH3 comprises SEQ ID NO: 8 (ARLKGELKDAFDI);CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN); CDRL2 comprises SEQ ID NO: 5(AASSLQS); and CDRL3 comprises SEQ ID NO: 10 (QQSYSTPLT). Arepresentative antibody having such heavy and light chain variable CDRregions is mAb24.

In each case, where specific sequences are recited, embodimentscomprising a sequence having at least 90% (e.g., at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical) identity tothe recited sequence (e.g., SEQ ID NO: 1-34 and 36-39) are alsoprovided.

The disclosure also provides, in some aspects, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises a heavy chainvariable region comprising an amino acid sequence that is at least 90%identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical) to SEQ ID NO: 35, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and a light chainvariable region comprising an amino acid sequence that is at least 90%identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical) to SEQ ID NO: 59, 60, 61, 62, or 63.

The disclosure also provides, in some aspects, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises a heavy chainvariable region comprising an amino acid sequence that is at least 90%identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical) to SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58 and a light chain variableregion comprising an amino acid sequence that is at least 90% identical(e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical) to SEQ ID NO: 59.

The disclosure also provides, in some aspects, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises a heavy chainvariable region comprising an amino acid sequence that is at least 90%identical (e.g., at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical) to SEQ ID NO: 35 and a light chain variableregion comprising an amino acid sequence that is at least 90% identical(e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical) to SEQ ID NO: 59, 60, 61, 62, or 63.

Antibodies mAb1-mAb23 are affinity matured antibodies derived fromparent antibody mAb24, as described in the Examples. An affinity maturedantibody or antigen-binding portion thereof is an antibody orantigen-binding fragment with one or more alterations (e.g., in one ormore CDRs or FRs) that result in an improvement in the affinity of anantibody for its antigen, compared to a parent antibody lacking thealteration(s). In some embodiments, an affinity matured antibody hasnanomolar or picomolar affinity for PD-L1. In some embodiments, thePD-L1 antibody or antigen-binding portion thereof has a K_(D) of atleast 1×10⁻⁷ M, at least 1×10⁻⁸ M, at least 1×10⁻⁹M, at least 1×10⁻¹⁰ M,at least 1×10⁻¹¹ M, at least 1×10⁻¹² M, or at least 1×10⁻¹³ M.

Table 1 shows the binding affinities (K_(D)) of mAb1, mAb2, mAb3, mAb4,mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12, mAb13, mAb14, mAb15,mAb16, mAb17, mAb18, mAb19, mAb20, mAb21, mAb22, and mAb23 (i.e.,affinity matured variants of mAb24) to human PD-L1. The term K_(D), asused herein, refers to the dissociation equilibrium constant of aparticular antibody-antigen interaction. K_(D)=k_(d)/k_(a). The termk_(d) (sec⁻¹), as used herein, refers to the dissociation rate constantof a particular antibody-antigen interaction. The value is also referredto as the k_(off) value. The term k_(a) (M⁻¹×sec⁻¹), as used herein,refers to the association rate constant of a particular antibody-antigeninteraction. The value is also referred to as the k_(on) value.

TABLE 1 Binding affinities of mAb1-mAb23 to human PD-L1 mAb K_(D) (nM)Fold Improvement 1 4.16 1.1 2 2.2 2.0 3 0.9 4.8 4 4.9 0.9 5 27.1 0.2 635.9 0.1 7 — — 8 3.7 1.2 9 5.4 0.8 10 6.3 0.7 11 156 — 12 358 — 13 52.30.1 14 — — 15 — — 16 — — 17 — — 18 527 — 19 817 — 20 53.9 0.1 21 91.50.1 22 2.5 1.8 23 369 —

Table 2 provides cell binding data for mAb1, mAb2, mAb3, mAb4, mAb5,mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12, mAb13, mAb14, mAb15, mAb16,mAb17, mAb18, mAb19, mAb20, mAb21, mAb22, and mAb23 (i.e., affinitymatured variants of mAb24) to human PD-L1 (“huPDL1”), cyno PD-L1, ormurine PD-L1 (“muPDL1”). Human or cyno PD-L1 was expressed on HEK cells;murine PD-L1 was expressed on A20 cells. Binding is expressed as an EC₅₀value, which can be estimated from titrating different concentrations ofmAb on cells that exogenously express the antigen of interest.Fluorescent tagged secondaries can be used to detect and quantify themAb binding. The data shown in Table 2 was fit to a 1:1 binding modelusing built-in functions in GRAPHPAD, which yielded the EC₅₀ value.

TABLE 2 Cell binding of mAb1-mAb23 to human, cyno, and murine PD-L1expressed on cells huPDL1 Fold cynoPDL1 Fold muPDL1 Fold EC₅₀ Improve-EC₅₀ Improve- EC₅₀ Improve- mAb (nM) ment (nM) ment (nM) ment 1 0.04 3.30.09 3.21 0.74 5.26 2 0.06 2.2 0.08 3.76 2.00 1.96 3 0.11 1.3 ND — 1.273.08 4 0.04 3.3 0.07 4.10 NB — 5 0.09 1.5 0.39 0.74 NB — 6 0.13 1.1 0.470.61 NB — 7 ND — NB — NB — 8 ND — 0.08 3.42 NB — 9 0.06 2.5 0.07 4.15 NB— 10 0.04 3.2 0.09 3.05 NB — 11 1.37 0.1 NB — NB — 12 1.23 0.1 NB — NB —13 1.43 1.0 ND — NB — 14 1.54 0.1 NB — NB — 15 ND — NB — NB — 16 NB — NB— NB — 17 0.76 0.2 NB — NB — 18 0.38 0.4 0.06 4.96 0.84 4.67 19 1.20 0.10.26 1.08 2.84 1.38 20 0.13 1.0 0.12 2.31 2.66 1.47 21 0.14 1.0 NB — NB— 22 0.18 0.8 0.32 0.89 NB — 23 0.83 0.2 NB — NB — *NB = no binding; ND= not determined; human and cyno PDL1 expressed on HEK cells; murinePDL1 expressed on A20 cells

The disclosure also provides an antibody or antigen-binding portionthereof that specifically binds PD-L1, wherein the antibody orantigen-binding portion thereof comprises a heavy chain variable regioncomprising an amino acid sequence that is at least 90% identical (forexample, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identical) to any one of SEQ ID NOs: 35, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58, and a lightchain variable region comprising an amino acid sequence that is at least90% identical (for example, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical) to any one of SEQ ID NO: 59, 60,61, 62, or 63. In some embodiments, the heavy chain variable regioncomprises an amino acid sequence that differs by 15 amino acids or less,14 amino acids or less, 13 amino acids or less, 12 amino acids or less,11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8amino acids or less, 7 amino acids or less, 6 amino acids or less, 5amino acids or less, 4 amino acids or less, 3 amino acids or less, 2amino acids or less, or 1 amino acid from any one of SEQ ID NOs: 35, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or58. In some embodiments, the light chain variable region comprises anamino acid sequence that differs by 15 amino acids or less, 14 aminoacids or less, 13 amino acids or less, 12 amino acids or less, 11 aminoacids or less, 10 amino acids or less, 9 amino acids or less, 8 aminoacids or less, 7 amino acids or less, 6 amino acids or less, 5 aminoacids or less, 4 amino acids or less, 3 amino acids or less, 2 aminoacids or less, or 1 amino acid from any one of SEQ ID NO: 59, 60, 61,62, or 63. Tables 3 and 4 provide the sequences for heavy chain variablesequences SEQ ID Nos: 35 and 40-58, and light chain variable sequencesSEQ ID Nos: 59-63, respectively.

TABLE 3 Heavy chain anti-PD-L1 variable sequences SEQ ID NO Sequence 35QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTMVTVSS 40QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS 41QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS 42QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPNFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKGAGDIWGQGTLVTVSS 43QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKFELKDAFDIWGQGTLVTVSS 44QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDEWGQGTLVTVSS 45QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTAST 46QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKNELKDAFDIWGQGTLVTVSS 47QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGVIPFLGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGILKDALDIWGQGTLVTVSS 48QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQDLEWMGGIIPIVGIANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS 49QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGEFKDAFDIWGQGTLVTVSS 50QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPLFGTAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS 51QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRINPILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFSIWGQGTLVTVSS 52QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGRIIPIFGTADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS 53QVQLVQSGAEVKKPGSSVKVSCKASGGKFSSYAISWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKCAFDIWGQGTLVTVSS 54QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVTVSS 55QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGAATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSS 56QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIVATANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVTVSS 57QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGKATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRKGELKDAFDIWGQGTLVTVSS 58QVQLVQSGAEVKKPGSSVKVSCKASGGPFRSHAVSWVRQAPGQGLEWMGGIIPVFGTATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKSELKDAFDIWGQGTLVTVSS

TABLE 4 Light chain variable sequences SEQ ID NO Sequence 59DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTPLTFGGGTKVEIK 60DIQMTQSPSSLSASVGDRVTITCRASQWISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTPLTFGGGTKVEIK 61DIQMTQSPSSLSASVGDRVTITCRASQQISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTPLTFGGGTKVEIK 62DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCFQSYSTPLTFGGGTKVEIK 63DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSYSTILTFGGGTKVEIK

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 35, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or58, and light chain CDRs of any of the light chain variable regions ofSEQ ID NOs: 59, 60, 61, 62, or 63.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 35, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or58, and light chain CDRs of any of the light chain variable regions ofSEQ ID NOs: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDRresidues are numbered according to Kabat.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 35, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or58, and light chain CDRs of any of the light chain variable regions ofSEQ ID NOs: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDRresidues are numbered according to Chothia.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 35, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or58, and light chain CDRs of any of the light chain variable regions ofSEQ ID NOs: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDRresidues are numbered according to MacCallum.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 35, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or58, and light chain CDRs of any of the light chain variable regions ofSEQ ID NOs: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDRresidues are numbered according to AbM.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 35, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or58, and light chain CDRs of any of the light chain variable regions ofSEQ ID NOs: 59, 60, 61, 62, or 63, wherein the heavy and light chain CDRresidues are numbered according to IMGT.

B. PD-1 Antagonists

In some aspects and embodiments, the disclosure provides for anti-PD-1antagonists. In some embodiments, the anti-PD-1 antagonist is any of theanti-PD-1 antibodies or antigen-binding molecules disclosed herein. Insome embodiments, the anti-PD-1 antibody or antigen-binding molecule isnot a part of a multispecific antigen-binding construct, i.e., theanti-PD-1 antibody or antigen-binding molecule is not a part of aprotein construct that binds to multiple epitopes. In some embodiments,the anti-PD-1 antibody or antigen-binding portion can be combined with adifferent antibody or antigen-binding portion to form a multispecificantigen-binding construct. In some embodiments, the multispecificantigen-binding construct is capable of binding an epitope on PD-1 andan epitope on another protein. In some embodiments, the epitope on theother protein is on PD-L1.

In some embodiments, any of the multispecific antigen-binding constructsdisclosed herein comprises a PD-1 antagonist. In some embodiments, thePD-1 antagonist is an “inhibitory receptor.” As used herein, an“inhibitory receptor” refers generally to an immune checkpoint moleculethat, when bound by a cognate ligand, causes suppression or inhibitionof an immune response, such as those known to enhance tumor evasion.However, in some instances as used herein, “inhibitory receptor” refersspecifically to PD-1.

PD-1 is an immune checkpoint inhibitory receptor that contains an“Immunoreceptor Tyrosine-based Inhibition Motif” or “ITIM”, comprising aconserved sequence of amino acids (S/I/V/L)xYxx(I/V/L) where x is anyamino acid. Methods for assaying whether PD-1 activity has beeninhibited are known in the art and can be readily designed by those ofskill in the art. Such assays include, for example, testing the effectsof any downstream signaling pathway(s) of PD-1 in vitro or in vivo.After PD-1 interacts with its ligand, the ITIM motif becomesphosphorylated by enzymes of, e.g., the Src kinase family, allowing themto recruit other enzymes, e.g., the phosphotyrosine phosphatases SHP-1and SHP-2, or the inositol-phosphatase called SHIP. These phosphataseshave been shown to decrease the activation of molecules involved in cellsignaling. See, e.g., Barrow & Trowsdale (2006) Eur J Immunol. 36 (7):1646-53. Thus, the phosphorylation state of the ITIM motif within PD-1can be assessed using known methods in the art. Also, the presence ofdownstream factors, such as phosphotyrosine phosphatases can also beexamined. Moreover, various cell-based assays and kits that detect thepresence of downstream factors (e.g., nuclear factor of activatedT-cells—NFAT—as a measure of PD-1 inhibition) as a proxy for PD-1activity state are known in the art. In other examples, simple bindingassays can be used to determine whether the construct of the presentdisclosure can block binding PD-1 and its ligand, as discussed above.

Accordingly, in some aspects, provided herein are antibodies orantigen-binding portions thereof that specifically bind PD-1. In someaspects, the antibody or antigen-binding portion thereof thatspecifically binds PD-1 comprises (a) a heavy chain variable regioncomprising (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX₁X₂YAX₃X₄, whereinX₁=S, R, G, or N; X₂=D, S, N, A, R, or G; X₃=M or L; X₄=S, L, or N);(ii) a CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a CDRH3comprising SEQ ID NO: 72 (ARGLDFIVGX₅TGNDY, wherein X₅=A, Y, or R); and(b) a light chain variable region comprising: (i) a CDRL1 comprising SEQID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS);and (iii) a CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 74(ARGLDFIVGATGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 74 (ARGLDFIVGATGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb25.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 75(ARGLDFIVGYTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 73 (FTFSDYAMS),CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb26.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 76 (FTFSSYAMS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 75(ARGLDFIVGYTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 76 (FTFSSYAMS),CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb27.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 77 (FTFSSYAML), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 75(ARGLDFIVGYTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 77 (FTFSSYAML),CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb28.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 78 (FTFSNYALS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 75(ARGLDFIVGYTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 78 (FTFSNYALS);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb29.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 79 (FTFSAYAMN), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 75(ARGLDFIVGYTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 79 (FTFSAYAMN);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb30.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 75(ARGLDFIVGYTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb31.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 81 (FTFGRYAMS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 75(ARGLDFIVGYTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 81 (FTFGRYAMS);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb32.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 82 (FTFNSYAMS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 75(ARGLDFIVGYTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 82 (FTFNSYAMS);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 75 (ARGLDFIVGYTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb33.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 83 (FTFSNYAMS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 74(ARGLDFIVGATGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 83 (FTFSNYAMS);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 74 (ARGLDFIVGATGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb34.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 84 (FTFSGYAMS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 85(ARGLDFIVGRTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 84 (FTFSGYAMS);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 85 (ARGLDFIVGRTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb35.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 86 (FTFSSYAMN), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 85(ARGLDFIVGRTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 86 (FTFSSYAMN);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 85 (ARGLDFIVGRTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb36.

In some embodiments of these aspects and all such aspects describedherein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS), CDRH2 comprises SEQID NO: 71 (SAISNSGTYTYYA) and CDRH3 comprises SEQ ID NO: 85(ARGLDFIVGRTGNDY). In some embodiments of these aspects and all suchaspects described herein, CDRH1 comprises SEQ ID NO: 80 (FTFRSYAMS);CDRH2 comprises SEQ ID NO: 71 (SAISNSGTYTYYA); CDRH3 comprises SEQ IDNO: 85 (ARGLDFIVGRTGNDY); CDRL1 comprises SEQ ID NO: 9 (RASQSISSYLN);CDRL2 comprises SEQ ID NO: 5 (AASSLQS); and CDRL3 comprises SEQ ID NO:10 (QQSYSTPLT). A representative antibody having such heavy and lightchain variable CDR regions is mAb37.

In each case, where specific sequences are recited, embodimentscomprising a sequence having at least 85%, (e.g., at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%identical) identity to the recited sequence (e.g., SEQ ID NO: 5, 9, 10,or 71-86) are also provided.

The disclosure also provides, in some aspects, an antibody orantigen-binding portion thereof that specifically binds PD-1, whereinthe antibody or antigen-binding portion thereof comprises a heavy chainvariable region comprising an amino acid sequence that is at least 85%,(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical) to any one of SEQ ID NO: 87-99, and alight chain variable region comprising an amino acid sequence that is atleast 85%, (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical) to SEQ ID NO: 59. In someembodiments, the heavy chain variable region comprises an amino acidsequence that is at least 85%, (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical) to SEQID NO: 90, and a light chain variable region comprising an amino acidsequence that is at least 85%, (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical) to SEQID NO: 59. Table 7 provides the sequences for heavy chain variablesequences SEQ ID Nos: 87-99, and Table 4 provides light chain variablesequence SEQ ID NO: 59, respectively. In some embodiments, the heavychain variable region comprises an amino acid sequence that differs by15 amino acids or less, 14 amino acids or less, 13 amino acids or less,12 amino acids or less, 11 amino acids or less, 10 amino acids or less,9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6amino acids or less, 5 amino acids or less, 4 amino acids or less, 3amino acids or less, 2 amino acids or less, or 1 amino acid from any oneof SEQ ID NOs: 87-99. In some embodiments, the light chain variableregion comprises an amino acid sequence that differs by 15 amino acidsor less, 14 amino acids or less, 13 amino acids or less, 12 amino acidsor less, 11 amino acids or less, 10 amino acids or less, 9 amino acidsor less, 8 amino acids or less, 7 amino acids or less, 6 amino acids orless, 5 amino acids or less, 4 amino acids or less, 3 amino acids orless, 2 amino acids or less, or 1 amino acid from SEQ ID NO: 59.

Antibodies mAb26-mAb37 are affinity matured antibodies derived fromparent antibody mAb25, as described in the Examples. An affinity maturedantibody or antigen-binding portion thereof is an antibody orantigen-binding fragment with one or more alterations (e.g., in one ormore CDRs or FRs) that result in an improvement in the affinity of anantibody for its antigen, compared to a parent antibody lacking thealteration(s). In some embodiments, an affinity matured antibody hasnanomolar or picomolar affinity for PD-1. In some embodiments, the PD-1antibody or antigen-binding portion thereof has a K_(D) of at least1×10⁻⁷M, at least 1×10⁻⁸M, at least 1×10⁻⁹M, at least 1×10⁻¹⁰ M, atleast 1×10⁻¹¹ M, at least 1×10⁻¹²M, or at least 1×10⁻¹³ M.

Tables 5 and 6 provide cell binding data for mAb25, mAb26, mAb27, mAb28,mAb29, mAb30, mAb31, mAb32, mAb33, mAb34, mAb35, mAb36, and mAb37 (i.e.,affinity matured variants of mAb25) to human PD-1 (“huPD-1”), cyno PD-1(“cyPD-1”), or murine PD-1 (“muPD-1”). Human, cyno, or murine PD-1 wasexpressed on CHO cells. Binding is expressed as an EC₅₀ value, which canbe estimated from titrating different concentrations of mAb on cellsthat exogenously express the antigen of interest. Fluorescent taggedsecondaries can be used to detect and quantify the mAb binding. The datashown in Table 5 was fit to a 1:1 binding model using built-in functionsin GRAPHPAD, which yielded the EC₅₀ value.

TABLE 5 Binding affinities of mAb25-mAb37 to human PD-1 K_(D) Foldimprovement mAb (nM) over Parent mAb25 240 mAb26 10 24 mAb27 2.3 104mAb28 5.6 43 mAb29 8.6 28 mAb30 3.1 77 mAb31 5.5 44 mAb32 2.1 114 mAb337 34 mAb34 7.2 33 mAb35 9 27 mAb36 7.7 31 mAb37 14 17

TABLE 6 Cell binding of mAb25-mAb37 to human, cyno, and murine PD-1expressed on cells huPD-1 Fold cyPD-1 Fold muPD-1 EC₅₀ improvement EC₅₀improvement EC₅₀ mAb (nM) over Parent (nM) over Parent (nM) mAb25 0.9158.59 NB mAb26 3.74 0.2 1.54 5.6 17.9 mAb27 1.75 0.5 1.53 5.6 6.14 mAb283.94 0.2 1.48 5.8 0.814 mAb29 1.27 0.7 1.73 5.0 2.14 mAb30 2.77 0.3 2.054.2 4.05 mAb31 1.71 0.5 1.9 4.5 1.87 mAb32 1.51 0.6 1.29 6.7 2.8 mAb331.04 0.9 1.33 6.5 10.4 mAb34 3.87 0.2 1.33 6.5 20.5 mAb35 1.64 0.6 2.663.2 PF mAb36 1.01 0.9 1.19 7.2 PF mAb37 0.75 1.2 1.09 7.9 0.942 *NB = nobinding; PF = poor fit; human, cyno, murine PD-1 expressed on CHO cells

TABLE 7 Heavy chain anti-PD-1 variable sequences SEQ ID NO Sequence 87EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGATGNDYWGQGTLVTVSS 88EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSS 89EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSS 90EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMLWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSS 91EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYALSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSS 92EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYAMNWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSS 93EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSS 94EVQLLESGGGLVQPGGSLRLSCAASGFTFGRYAMSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSS 95EVQLLESGGGLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSS 96EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGATGNDYWGQGTLVTVSS 97EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGRTGNDYWGQGTLVTVSS 98EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGRTGNDYWGQGTLVTVSS 99EVQLLESGGGLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGRTGNDYWGQGTLVTVSS

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and light chain CDRs ofthe light chain variable region of SEQ ID NO: 59.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and light chain CDRs ofthe light chain variable region of SEQ ID NO: 59, wherein the heavy andlight chain CDR residues are numbered according to Kabat.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and light chain CDRs ofthe light chain variable region of SEQ ID NO: 59, wherein the heavy andlight chain CDR residues are numbered according to Chothia.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and light chain CDRs ofthe light chain variable region of SEQ ID NO: 59, wherein the heavy andlight chain CDR residues are numbered according to MacCallum.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and light chain CDRs ofthe light chain variable region of SEQ ID NO: 59, wherein the heavy andlight chain CDR residues are numbered according to AbM.

The disclosure also provides, in some embodiments, an antibody orantigen-binding portion thereof that specifically binds PD-L1, whereinthe antibody or antigen-binding portion thereof comprises heavy chainCDRs of any of the heavy chain variable regions of SEQ ID NOs: 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99, and light chain CDRs ofthe light chain variable region of SEQ ID NO: 59, wherein the heavy andlight chain CDR residues are numbered according to IMGT.

C. Multispecific Antigen-Binding Constructs

The present disclosure provides, in some aspects, compositions andmethods for enhancing an immune response to tumor cells by inhibitingthe interaction between PD-L1 and PD-1, for example, the interactionbetween PD-L1 expressed on a tumor cell and PD-1 expressed on a T cell.Antibodies or antigen-binding portions thereof that specifically orselectively bind PD-L1 or PD-1 are provided. As used herein, the terms“specifically binds to,” “specific for,” “selectively binds” and“selective for” PD-L1 or PD-1, or an epitope on PD-L1 or PD-1, meanbinding that is measurably different from a non-specific ornon-selective interaction. In some embodiments, the antibody orantigen-binding portion thereof specifically binds to human PD-L1 orPD-1 and/or mouse PD-L1 or PD-1. Specific binding can be measured, forexample, by determining binding of a molecule compared to binding of acontrol molecule. Specific binding can also be determined by competitionwith a control molecule that is similar to the target, such as an excessof non-labeled target. In that case, specific binding is indicated ifthe binding of the labeled target to a probe is competitively inhibitedby the excess non-labeled target.

In some embodiments, any of the multispecific antigen-binding constructsdisclosed herein binds to at least two different receptors or epitopes(e.g., PD-1 and PD-L1), wherein the two different receptors or epitopesbound by the multispecific antigen-binding construct are expressed onthe surface of the same cell. For example, in some embodiments, themultispecific antigen-binding construct simultaneously binds to PD-1 andPD-L1, wherein the PD-1 and PD-L1 are expressed on the surface of thesame cell. In some embodiments, any of the multispecific antigen-bindingconstructs disclosed herein binds to at least two different receptors orepitopes (e.g., PD-1 and PD-L1), wherein the two different receptors orepitopes bound by the multispecific antigen-binding construct areexpressed on the surface of two different cells. For example, in someembodiments, the multispecific antigen-binding construct simultaneouslybinds to PD-1 expressed on the surface of a first cell and to PD-L1expressed on the surface of a second cell.

In some embodiments, the multispecific antigen-binding construct iscapable of binding human PD-1. In some embodiments, the multispecificantigen-binding construct is capable of binding murine PD-1. In someembodiments, the multispecific antigen-binding construct is capable ofbinding cynomolgus monkey PD-1. In some embodiments, the multispecificantigen-binding construct is capable of binding human, murine, andcynomolgus monkey PD-1 with similar affinity.

In some aspects and embodiments, the disclosure provides for amultispecific antigen-binding construct comprising at least two units ofantigen-binding, wherein a first unit of antigen-binding binds PD-1, anda second unit of antigen-binding binds a PD-1 ligand. In someembodiments, the first unit of antigen-binding binds PD-1 expressed byan immune cell. In some embodiments, the second unit of antigen-bindingbinds PD-1 expressed by a second cell. In some embodiments, themultispecific antigen-binding construct blocks the interaction of PD-1and a PD-1 ligand, such as PD-L1 or PD-L2. In some embodiments, themultispecific antigen-binding construct blocks the interaction of PD-1and a PD-1 ligand, such as PD-L1 or PD-L2. In some embodiments, themultispecific antigen-binding construct comprises at least two units ofantigen-binding that bind PD-1. In some embodiments, the multispecificantigen-binding construct comprises two units of antigen-binding thatbind PD-1. In some embodiments, the multispecific antigen-bindingconstruct comprises at least two units of antigen-binding that bind aPD-1 ligand, such as PD-L1 or PD-L2. In some embodiments, themultispecific antigen-binding construct comprises two units ofantigen-binding that bind a PD-1 ligand, such as PD-L1 or PD-L2. In someembodiments, the multispecific antigen-binding construct comprises atleast four units of antigen-binding, wherein two units ofantigen-binding bind PD-1 and two units of antigen-binding bind a PD-1ligand, such as PD-L1 or PD-L2. In some embodiments, the multispecificantigen-binding construct comprises four units of antigen-binding,wherein two units of antigen-binding bind PD-1 and two units ofantigen-binding bind a PD-1 ligand, such as PD-L1 or PD-L2. In someembodiments, each unit of antigen-binding is capable of bindingindependently to its cognate antigen, i.e., PD-1 or a PD-1 ligand, suchas PD-L1 or PD-L2. In some embodiments, the multispecificantigen-binding construct promotes loss of PD-1 expression from a cell.In some embodiments, the loss of PD-1 expression is due to PD-1shedding. In some embodiments, the multispecific antigen-bindingconstruct blocks interaction of PD-1 and the PD-1 ligand, such as PD-L1or PD-L2. In some embodiments, the multispecific antigen-bindingconstruct comprises a common light chain. For example, at least twounits of antigen-binding comprise a common light chain.

In some embodiments, the first unit of antigen-binding binds PD-1 andcomprises:

-   -   (a) a heavy chain variable region comprising (i) a CDRH1        comprising SEQ ID NO: 70 (FTFX₁X₂YAX₃X₄, wherein X₁=S, R, G, or        N; X₂=D, S, N, A, R, or G; X₃=M or L; X₄=S, L, or N); (ii) a        CDRH2 comprising SEQ ID NO: 71 (SAISNSGTYTYYA); and (iii) a        CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX₅TGNDY, wherein X₅=A,        Y, or R); and    -   (b) a light chain variable region comprising: (i) a CDRL1        comprising SEQ ID NO: 9 (RASQSISSYLN); (ii) a CDRL2 comprising        SEQ ID NO: 5 (AASSLQS); and (iii) a CDRL3 comprising SEQ ID NO:        10 (QQSYSTPLT).

In some such embodiments, the first unit of antigen-binding binds PD-1and comprises:

-   -   (a) a CDRH1 comprising SEQ ID NO: 73 (FTFSDYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 74 (ARGLDFIVGATGNDY);    -   (b) a CDRH1 comprising SEQ ID NO: 73 (FTFSDYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (c) a CDRH1 comprising SEQ ID NO: 76 (FTFSSYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (d) a CDRH1 comprising SEQ ID NO: 77 (FTFSSYAML), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (e) a CDRH1 comprising SEQ ID NO: 78 (FTFSNYALS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (f) a CDRH1 comprising SEQ ID NO: 79 (FTFSAYAMN), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (g) a CDRH1 comprising SEQ ID NO: 80 (FTFRSYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (h) a CDRH1 comprising SEQ ID NO: 81 (FTFGRYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (i) a CDRH1 comprising SEQ ID NO: 82 (FTFNSYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 75 (ARGLDFIVGYTGNDY);    -   (j) a CDRH1 comprising SEQ ID NO: 83 (FTFSNYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 74 (ARGLDFIVGATGNDY);    -   (k) a CDRH1 comprising SEQ ID NO: 84 (FTFSGYAMS), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising        SEQ ID NO: 85 (ARGLDFIVGRTGNDY);    -   (l) a CDRH1 comprising SEQ ID NO: 86 (FTFSSYAMN), a CDRH2        comprising SEQ ID NO: 71 (SAISNSGTYTYYA) and a CDRH3 comprising        SEQ ID NO: 85 (ARGLDFIVGRTGNDY); or    -   (m) a CDRH1 comprising SEQ ID NO: 80 (FTFRSYAMS), a comprising        SEQ ID NO: 71 (SAISNSGTYTYYA), and a CDRH3 comprising SEQ ID NO:        85 (ARGLDFIVGRTGNDY).

In some embodiments, the first unit of antigen-binding binds PD-1 andcomprises:

-   -   (a) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 87;    -   (b) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 88;    -   (c) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 89;    -   (d) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 90;    -   (e) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 91;    -   (f) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 92;    -   (g) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 93;    -   (h) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 94;    -   (i) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 95;    -   (j) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 96;    -   (k) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 97;    -   (l) a heavy chain variable region comprising an amino acid        sequence that is at least 90% identical to SEQ ID NO: 98; or    -   (m) a heavy chain variable region comprising amino acid sequence        that is at least 90% identical to SEQ ID NO: 99.

In some embodiments, the first unit of antigen-binding binds PD-1 andcomprises a light chain variable region comprising an amino acidsequence that is at least 90% identical to SEQ ID NO: 59.

In some embodiments, the second unit of antigen-binding binds PD-L2. Insome embodiments, the second unit of antigen-binding binds PD-L1. Insome embodiments, the second unit of antigen-binding binds PD-L1 andcomprises:

-   -   a. a heavy chain variable region comprising (i) a CDRH1        comprising SEQ ID NO: 1 (GTFSSYAIN); (ii) a CDRH2 comprising SEQ        ID NO: 2 (GGIIPX₁X₂GX₃ATYA, wherein X₁ is V or I; X₂ is F, L, or        V; and X₃ is T or A); and (iii) a CDRH3 comprising SEQ ID NO: 3        (ARLKX₁ELKDAFDI, wherein X₁ is G, F, or N); and    -   b. a light chain variable region comprising: (i) a CDRL1        comprising SEQ ID NO: 4 (RASQX₁ISSYLN, wherein X₁ is S, W, or        Q); (ii) a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and (iii) a        CDRL3 comprising SEQ ID NO: 6 (X₁QSYSTPLT, wherein X₁ is Q or        F).

In some such embodiments, the second unit of antigen-binding binds PD-L1and comprises:

-   -   (a) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 7 (GGIIPILGAATYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (b) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 7 (GGIIPILGAATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (c) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (d) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA) and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (e) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 12        (RASQWISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (f) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 13        (RASQQISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (g) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (h) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 15 (GGIIPIFGIANYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (i) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 15 (GGIIPIFGIANYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (j) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 16 (GGIIPNFGTATYA), and a CDRH3 comprising        SEQ ID NO: 17 (ARLKGELKGAGDI);    -   (k) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 16 (GGIIPNFGTATYA), a CDRH3 comprising SEQ        ID NO: 17 (ARLKGELKGAGDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (l) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 18 (ARLKFELKDAFDI);    -   (m) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 18 (ARLKFELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (n) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 19 (ARLKGELKDAFDE);    -   (o) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 19 (ARLKGELKDAFDE), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (p) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 20 (ARLKNELKDAFDI);    -   (q) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 20 (ARLKNELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (r) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 21 (GGVIPFLGTANYA), and a CDRH3 comprising        SEQ ID NO: 22 (ARLKGILKDALDI);    -   (s) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 21 (GGVIPFLGTANYA), a CDRH3 comprising SEQ        ID NO: 22 (ARLKGILKDALDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (t) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising EQ ID NO: 29 (GRIIPIFGTADYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (u) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 29 (GRIIPIFGTADYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (v) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 31 (GGIIPILGTATYA), and a CDRH3 comprising        SEQ ID NO: 32 (ARRKGELKDAFDI);    -   (w) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 31 (GGIIPILGTATYA), a CDRH3 comprising SEQ        ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (x) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 33 (GGIIPIVATANYA), and a CDRH3 comprising        SEQ ID NO: 32 (ARRKGELKDAFDI);    -   (y) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 33 (GGIIPIVATANYA), a CDRH3 comprising SEQ        ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (z) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 34 (GGIIPIFGKATYA), and a CDRH3 comprising        SEQ ID NO: 32 (ARRKGELKDAFDI);    -   (aa) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 34 (GGIIPIFGKATYA), a CDRH3 comprising SEQ        ID NO: 32 (ARRKGELKDAFDI), a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (bb) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 38 (FQSYSTPLT);    -   (cc) a CDRH1 comprising SEQ ID NO: 1 (GTFSSYAIN), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), a CDRH3 comprising SEQ        ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 39 (QQSYSTILT);    -   (dd) a CDRH1 comprising SEQ ID NO: 14 (GTFSSYAFS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA) and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (ee) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 24 (GGIIPIVGIANYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (ff) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 24 (GGIIPIVGIANYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (gg) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 25 (ARLKGEFKDAFDI);    -   (hh) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 25 (ARLKGEFKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (ii) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI);    -   (jj) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 26 (GRIIPLFGTAHYA), and a CDRH3 comprising        SEQ ID NO: 8 (ARLKGELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (kk) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 27 (GRINPILGTANYA), and a CDRH3 comprising        SEQ ID NO: 28 (ARLKGELKDAFSI);    -   (ll) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 27 (GRINPILGTANYA), and a CDRH3 comprising        SEQ ID NO: 28 (ARLKGELKDAFSI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (mm) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 30 (ARLKGELKCAFDI);    -   (nn) a CDRH1 comprising SEQ ID NO: 23 (GTFSSYAIS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a comprising SEQ        ID NO: 30 (ARLKGELKCAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT);    -   (oo) a CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 37 (ARLKSELKDAFDI); or    -   (pp) a CDRH1 comprising SEQ ID NO: 36 (GPFRSHAVS), a CDRH2        comprising SEQ ID NO: 11 (GGIIPVFGTATYA), and a CDRH3 comprising        SEQ ID NO: 37 (ARLKSELKDAFDI); a CDRL1 comprising SEQ ID NO: 9        (RASQSISSYLN); a CDRL2 comprising SEQ ID NO: 5 (AASSLQS); and a        CDRL3 comprising SEQ ID NO: 10 (QQSYSTPLT).

In some such embodiments, the second unit of antigen-binding binds PD-L1and comprises a heavy chain variable region comprising an amino acidsequence that is at least 90% identical to any one of SEQ ID NOs: 35,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,or 58, and a light chain variable region comprising an amino acidsequence that is at least 90% identical to any one of SEQ ID NOs: 59,60, 61, 62, or 63.

In some embodiments, the second unit of antigen-binding binds PD-L1 andcomprises a heavy chain variable region comprising:

(a) an amino acid sequence that is at least 90% identical to SEQ ID NO:35;

(b) an amino acid sequence that is at least 90% identical to SEQ ID NO:40;

(c) an amino acid sequence that is at least 90% identical to SEQ ID NO:41;

(d) an amino acid sequence that is at least 90% identical to SEQ ID NO:42;

(e) an amino acid sequence that is at least 90% identical to SEQ ID NO:43;

(f) an amino acid sequence that is at least 90% identical to SEQ ID NO:44;

(g) an amino acid sequence that is at least 90% identical to SEQ ID NO:45;

(h) an amino acid sequence that is at least 90% identical to SEQ ID NO:46;

(i) an amino acid sequence that is at least 90% identical to SEQ ID NO:47;

(j) an amino acid sequence that is at least 90% identical to SEQ ID NO:48;

(k) an amino acid sequence that is at least 90% identical to SEQ ID NO:49;

(l) an amino acid sequence that is at least 90% identical to SEQ ID NO:50;

(m) an amino acid sequence that is at least 90% identical to SEQ ID NO:51;

(n) an amino acid sequence that is at least 90% identical to SEQ ID NO:52;

(o) an amino acid sequence that is at least 90% identical to SEQ ID NO:53;

(p) an amino acid sequence that is at least 90% identical to SEQ ID NO:54;

(q) an amino acid sequence that is at least 90% identical to SEQ ID NO:55;

(r) an amino acid sequence that is at least 90% identical to SEQ ID NO:56;

(s) an amino acid sequence that is at least 90% identical to SEQ ID NO:57; or

(t) an amino acid sequence that is at least 90% identical to SEQ ID NO:58;

In some embodiments, the second unit of antigen-binding binds PD-L1 andcomprises a light chain variable region comprising:

(a) an amino acid sequence that is at least 90% identical to SEQ ID NO:59;

(b) an amino acid sequence that is at least 90% identical to SEQ ID NO:60;

(c) an amino acid sequence that is at least 90% identical to SEQ ID NO:61;

(d) an amino acid sequence that is at least 90% identical to SEQ ID NO:62; or

(e) an amino acid sequence that is at least 90% identical to SEQ ID NO:63.

In some embodiments, the disclosure provides for a multispecificantigen-binding construct comprising any of the PD-1 antagonistsdisclosed herein and any of the antagonists of a PD-1 ligand, such asPD-L1, disclosed herein. For example, Bispecific 3 is a multispecific,tetravalent antigen-binding construct that specifically binds human PD-1and human PD-L1. The construct comprises an anti-PD-1 IgG1 antibody(mAb28) in which the heavy chain of the antibody is a fusion proteinfurther comprising at its C-terminus the heavy chain variable region ofan anti-PD-L1 antibody (mAb1), which is connected to the Fc region ofthe anti-BCMA antibody by way of a poly-GGGS (SEQ ID NO: 120) linker.The light chains for the anti-PD-1 portion and the anti-PD-L1 portionsof the construct are identical (SEQ ID NO: 101). Bispecific 3, thestructure for which is represented by the illustration in FIG. 13A,comprises the heavy chain sequence recited in SEQ ID NO: 100 and thelight chain sequence recited in SEQ ID NO: 101.

In some embodiments, the disclosure provides for a multispecificantibody or antigen-binding portion thereof that specifically binds PD-1and PD-L1, wherein the antibody or antigen-binding portion thereofcomprises a heavy chain region comprising an amino acid sequence that isat least 85%, (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical) to SEQ ID NO: 100 or 102and a light chain region comprising an amino acid sequence that is atleast 85%, (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical) to SEQ ID NO: 101 or 103.

In some embodiments, the disclosure provides for a multispecificantibody or antigen-binding portion thereof that specifically binds PD-1and PD-L1, wherein the antibody or antigen-binding portion thereofcomprises a heavy chain region comprising an amino acid sequence that isat least 85%, (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical) to SEQ ID NO: 100, and alight chain region comprising an amino acid sequence that is at least85%, (e.g., at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical) to SEQ ID NO: 101. In someembodiments, the heavy chain region comprises an amino acid sequencethat differs by 15 amino acids or less, 14 amino acids or less, 13 aminoacids or less, 12 amino acids or less, 11 amino acids or less, 10 aminoacids or less, 9 amino acids or less, 8 amino acids or less, 7 aminoacids or less, 6 amino acids or less, 5 amino acids or less, 4 aminoacids or less, 3 amino acids or less, 2 amino acids or less, or 1 aminoacid from SEQ ID NO: 100. In some embodiments, the light chain regioncomprises an amino acid sequence that differs by 15 amino acids or less,14 amino acids or less, 13 amino acids or less, 12 amino acids or less,11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8amino acids or less, 7 amino acids or less, 6 amino acids or less, 5amino acids or less, 4 amino acids or less, 3 amino acids or less, 2amino acids or less, or 1 amino acid from SEQ ID NO: 101.

In some embodiments, the disclosure provides for a multispecificantibody or antigen-binding portion thereof that specifically binds PD-1and PD-L1, wherein the antibody or antigen-binding portion thereofcomprises a heavy chain region comprising an amino acid sequence that isat least 85%, (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical) to SEQ ID NO: 102, and alight chain region comprising an amino acid sequence that is at least85%, (e.g., at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical) to SEQ ID NO: 103. In someembodiments, the heavy chain region comprises an amino acid sequencethat differs by 15 amino acids or less, 14 amino acids or less, 13 aminoacids or less, 12 amino acids or less, 11 amino acids or less, 10 aminoacids or less, 9 amino acids or less, 8 amino acids or less, 7 aminoacids or less, 6 amino acids or less, 5 amino acids or less, 4 aminoacids or less, 3 amino acids or less, 2 amino acids or less, or 1 aminoacid from SEQ ID NO: 102. In some embodiments, the light chain regioncomprises an amino acid sequence that differs by 15 amino acids or less,14 amino acids or less, 13 amino acids or less, 12 amino acids or less,11 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8amino acids or less, 7 amino acids or less, 6 amino acids or less, 5amino acids or less, 4 amino acids or less, 3 amino acids or less, 2amino acids or less, or 1 amino acid from SEQ ID NO: 103.

Also provided herein, in some aspects and embodiments, is amultispecific antigen-binding construct comprising four units ofantigen-binding, wherein two units of antigen-binding bind PD-1 and twounits of antigen-binding bind PD-L1, and wherein the construct comprisesa heavy chain amino acid sequence that is at least 85%, identical to theamino acid sequence of SEQ ID NO: 100 or 102, and a light chain aminoacid sequence that is at least 85%, identical to the amino acid sequenceof SEQ ID NO: 101 or 103.

Also provided herein, in some aspects and embodiments, is amultispecific antigen-binding construct comprising four units ofantigen-binding, wherein two units of antigen-binding bind PD-1 and twounits of antigen-binding bind PD-L1, and wherein the construct comprisesa heavy chain amino acid sequence that is at least 85%, identical to theamino acid sequence of SEQ ID NO: 100 and a light chain amino acidsequence that is at least 85%, identical to the amino acid sequence ofSEQ ID NO: 101.

Also provided herein, in some aspects and embodiments, is amultispecific antigen-binding construct comprising four units ofantigen-binding, wherein two units of antigen-binding bind PD-1 and twounits of antigen-binding bind PD-L1, and wherein the construct comprisesa heavy chain amino acid sequence that is at least 85%, identical to theamino acid sequence of SEQ ID NO: 102 and a light chain amino acidsequence that is at least 85%, identical to the amino acid sequence ofSEQ ID NO: 103.

TABLE 8 Multispecific Heavy Chain Sequences SEQ ID NO Sequence 100EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMLWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGAATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT 102EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMLWVRQAPGKGLEWVSAISNSGTYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGLDFIVGYTGNDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFASTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPILGAATYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLKGELKDAFDIWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRP SETVTCNVAHPASSTKVDKKIVPRDCG

TABLE 9 Multispecific Light Chain Sequences SEQ ID NO Sequence 101DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 103DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

The term “multispecific antigen-binding construct”, as used hereinrefers to bispecific, tri-specific, or multispecific antigen-bindingconstructs, and antigen-binding portions or fragments thereof. Amultispecific antigen-binding construct can be a single multifunctionalpolypeptide, or it can be a multimeric complex of two or more molecules(e.g., polypeptides and/or aptamers) that are covalently ornon-covalently associated with one another. The term “multispecificantigen-binding constructs” includes antibodies (or antigen-bindingfragments thereof) that may be linked to or co-expressed with anotherfunctional molecule, e.g., another peptide, protein, and/or aptamer. Forexample, an antibody or fragment thereof can be functionally linked(e.g., by chemical coupling, genetic fusion, non-covalent association orotherwise) to one or more other molecular entities, such as a protein orfragment thereof to produce a bispecific or a multispecificantigen-binding molecule with a second binding specificity. As usedherein, the term “multispecific antigen-binding constructs” alsoincludes bispecific, trispecific or multispecific antibodies orantigen-binding fragments thereof. In certain embodiments, an antibodyis functionally linked to another antibody or antigen-binding fragmentthereof to produce a bispecific antibody with a second bindingspecificity. Bispecific and multispecific antibodies of the presentinvention are described elsewhere herein.

As used herein, an antigen-binding “arm” refers to a unit, domain,region, or the like, of the multispecific antigen-binding construct thatforms an area of the construct that binds to an antigen. Thus, a “firstarm” forms a separate binding area of the multispecific antigen-bindingconstruct from a “second arm” of the construct, each arm forming a unitof antigen binding. Generally, one “arm” (first arm) is distinct fromthe other “arm” (second arm) in its antigen binding or antigenspecificity. Thus, in the example of a bispecific, bivalent antibody,one arm of the antibody binds to antigen A, while the other arm of theantibody binds to antigen B. In some embodiments, in the example of abispecific, bivalent antibody, one arm of the antibody binds to antigenA, while the other arm of the antibody binds to antigen B or C(cross-reacts with two antigens such as PD-L1 and PD-L2 due to, e.g.,similarity in structure). See, e.g., U.S. Pat. No. 9,845,356. Similarly,in the example of a tetravalent bispecific antibody (formed, e.g., byjoining two different antibodies), one “arm” refers to the area of theantibody that binds to antigen A (even if two binding sites—of abivalent antibody—bind to antigen A) and the “other arm” refers to thearea of the antibody that binds to antigen B (even if two bindingsites—of a bivalent antibody—bind to antigen B). In some embodiments, inthe example of a tetravalent bispecific antibody (formed, e.g., byjoining two different antibodies), one “arm” refers to the area of theantibody that binds to antigen A (even if two binding sites—of abivalent antibody—bind to antigen A) and the “other arm” refers to thearea of the antibody that binds to antigen B or C (even if two bindingsites—of a bivalent antibody—can bind to antigen B or C). See, e.g.,U.S. Pat. No. 9,845,356. As would be apparent to those of skill in theart, “first” or “second” can be used interchangeably.

The term “valency,” when used to describe an antigen-binding constructor protein or antigen-binding arm, refers to the number of recognition(binding) sites in the antigen-binding construct or protein, regardlessof whether those different recognition or binding sites bind to the sameepitope. Each recognition site specifically recognizes, and is thereforecapable of binding, one epitope (binding site) on an antigen. When anantigen-binding protein comprises more than one recognition site (e.g.,when an antigen-binding protein is an IgG, which has two recognitionsites in its variable regions), each recognition site can specificallyrecognize the same epitope on the same antigen, or different epitopes,whether on the same or different antigens. Multivalency can increase theavidity, i.e., the strength of binding between an antigen-binding arm orconstruct and the pertinent antigen or target receptor. Avidity isrelated to both the affinity between an epitope or antigenic determinantand its binding site on the antigen-binding unit, and the actual numberof pertinent binding sites present on the antigen-binding unit.

In some embodiments, any of the multispecific antigen-binding constructsdisclosed herein comprises a multivalent (e.g., bivalent) antibody orantigen-binding fragment, wherein at least two of the valenciesspecifically bind PD-1. In some embodiments, any of the multispecificantigen-binding constructs disclosed herein comprises a multivalent(e.g., bivalent) antibody or antigen-binding fragment, wherein at leasttwo of the valencies specifically bind a PD-1 ligand (e.g., PD-L1 orPD-L2). In some embodiments, any of the multispecific antigen-bindingconstructs disclosed herein comprises a multivalent (e.g., bivalent)antibody or antigen-binding fragment, wherein at least two of thevalencies specifically bind PD-L1. In some embodiments, any of themultispecific antigen-binding constructs disclosed herein comprises afirst multivalent (e.g., bivalent) antibody or antigen-binding fragmentand a second multivalent (e.g., bivalent) antibody or antigen-bindingfragment, wherein at least two of the valencies of the first multivalentantibody or antigen-binding fragment specifically bind PD-1, and whereinat least two of the valencies of the second multivalent antibody orantigen-binding fragment specifically bind PD-L1. In some embodiments,any of the multispecific antigen-binding constructs disclosed herein isa tetravalent construct, wherein the tetravalent construct comprises afirst bivalent antibody or antigen-binding fragment and a secondbivalent antibody or antigen-binding fragment, wherein both valencies ofthe first bivalent antibody or antigen-binding fragment are specific forthe same epitope on PD-1, and wherein both valencies of the secondbivalent antibody or antigen-binding fragment are specific for the sameepitope on PD-L1. In some embodiments of such tetravalent constructs,the first and second bivalent antibody or antigen-binding fragment orportion thereof use a light chain having the same amino acid sequence.In other words, the tetravalent construct comprises a common lightchain. For example, a light chain having the sequence of SEQ ID NO: 101or SEQ ID NO: 103.

In some embodiments, the first arm is an antagonist of PD-1. In someembodiments, the second arm is an antagonist of a PD-1 ligand—e.g.,PD-L1 and/or PD-L2. In some embodiments, the first arm is an antagonistof PD-1, and the second arm is an antagonist of cognate PD-1ligand—e.g., PD-L1 and/or PD-L2.

The terms “antagonist, “antagonize,” and “inhibit” when used to refer tothe biological activity of the antigen-binding arm indicate that theantigen-binding arm binds its target (e.g., PD-1) on the respective celland partially or fully blocks, inhibits, and/or reduces the biologicalresponse through PD-1. In some embodiments, inhibition in the presenceof the antagonist is observed in a dose-dependent manner. In someembodiments, the measured signal (e.g., biological activity) is at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 100% lower than the signal measuredwith a negative control under comparable conditions. Also disclosedherein, are methods of identifying antagonists suitable for use in themethods of the disclosure. For example, these methods include, but arenot limited to, binding assays such as enzyme-linked immuno-absorbentassay (ELISA), FORTE BIO© systems, and radioimmunoassay (RIA). Theseassays determine the ability of an antagonist to bind the polypeptide ofinterest (e.g., PD-1 or its ligand) and therefore indicate the abilityof the antagonist to inhibit, neutralize or block the activity of thepolypeptide of interest. Efficacy of an antagonist can also bedetermined using functional assays, such as the ability of an antagonistto inhibit the function of the polypeptide. For example, a functionalassay may comprise contacting a polypeptide with a candidate antagonistmolecule and measuring a detectable change in one or more biologicalactivities normally associated with the polypeptide. The potency of anantagonist is usually defined by its IC₅₀ value (concentration requiredto inhibit 50% of the agonist response). The lower the IC₅₀ value thegreater the potency of the antagonist and the lower the concentrationthat is required to inhibit the maximum biological response.

In some embodiments, at least one antigen-binding arm has a K_(D) of atleast 1×10⁻⁷ M, at least 1×10⁻⁸M, at least 1×10⁻⁹M, at least 1×10⁻¹⁰ M,at least 1×10⁻¹¹M, at least 1×10⁻¹² M, or at least 1×10⁻¹³ M. In someembodiments, both antigen-binding arms have the same or similar K_(D).The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular antigen-binding arm/antigeninteraction. K_(D)=k_(d)/k_(a). The term “k_(a)” (sec⁻¹), as usedherein, refers to the dissociation rate constant of a particularantigen-binding arm/antigen interaction. This value is also referred toas the k_(off) value. The term “k_(a)” (M⁻¹×sec⁻¹), as used herein,refers to the association rate constant of a particular antigen-bindingarm/antigen interaction. This value is also referred to as the k_(on)value.

In some embodiments, the binding of one arm (e.g., the first arm) of themultispecific antigen-binding construct to its target does not block thebinding of the other arm (e.g., the second arm) to its target. In someembodiments, the binding of one arm does not sterically hinder thesecond arm from binding its target. For example, upon the binding of afirst arm to PD-1, the second arm is free to bind a ligand of PD-1(e.g., PD-L1 and/or PD-L2). Thus, in some embodiments, the first arm andsecond arm bind to their respective targets and both arms remain boundconcurrently.

In some embodiments, binding of the first arm and the second arm totheir respective targets bridges the immune cell and the second celltogether, bringing the two cells in closeproximity. As used herein,“bridge” refers to the joining of two cell types (e.g., one immune cellthat expresses PD-1, and a second cell that expresses its ligand—PD-L1),or bringing of the two cells together in close proximity; the two cellsneed not be in physical contact. Thus, the multispecific antigen-bindingconstruct acts as a connecter (e.g., a bridge) to the two cells, eachone expressing PD-1 or either of its ligands.

Methods for determining whether two cells are bridged or connectedtogether by a construct of the present invention are known in the art.For example, in some embodiments, the bridging of the immune cell andthe second cell is determined by, e.g., flow cytometry, FRET,immunoprecipitation, microscopy, or fluorescence plate reader.

In some embodiments, binding of the first arm and the second arm of amultispecific construct to their respective targets results indown-regulation and/or shedding of the ectodomain and/or degradation ofa target, e.g., PD-1. As used herein, “down-regulation” refers to theprocess by which a cell decreases the quantity of a cellular component,such as RNA or protein. In the case of cell-surface protein receptors,down-regulation can occur through internalization of the receptor as aconsequence of binding to a ligand or any of the constructs describedherein. Shedding or ectodomain shedding refers to a process by whichcell surface proteins are proteolytically cleaved resulting in therelease of their ectodomains into the extracellular milieu. Non-limitingexamples of sheddases that regulate ectodomain shedding include membersof the disintigrin and metalloproteinase (ADAM) family, such as ADAM8,ADAM9, ADAM 10, ADAM12, ADAM15, ADAM 17, and ADAM 28, and matrixmetalloproteinases (MMPs), such as MMP2, MMP3, MMP7, MMP9, and MMP14. Itis believed that the distance from the plasma membrane and structure ofthe cleavage site region are more important than the specific sequencein ectodomain shedding. Protein degradation or proteolysis refers to aset of processes that result in the hydrolysis of one or more of thepeptide bonds in a protein, either through catalysis by proteolyticenzymes called proteases, or nonenzymatically, for example at very lowor very high pH. In eukaryotic cells, two major pathways—theubiquitin-proteasome pathway and lysosomal proteolysis—mediate proteindegradation. Methods for determining whether a target receptor isdown-regulated and/or shedded and/or degraded by a multispecificconstruct disclosed herein are known in the art, and are described inthe Examples, see, for example, FIGS. 12A-12C; e.g., flow cytometry,Western blotting, immunoprecipitation, microscopy, or fluorescence platereader.

As described herein, the constructs of the present invention can bridgean immune cell that expresses PD-1, and a second cell that expresses itsligand, such as a second immune cell, and/or a cancer or tumor cell. Asthose of skill in the art would recognize, the type of immune celldepends on the context of the disease to be treated; the particular typeof immune cell can be readily determined depending on the disorder underconsideration. In some embodiments, the immune cell is a T cell, e.g.,regulatory T cells (a.k.a. suppressor T cells), including CD8+ T cellsand CD4+ T cells, and subtypes, such as CD4⁺FOXP3⁺ T_(reg) cells,CD4⁺FOXP3⁻ T_(reg) cells, Tr1 cells, Th3 cells, and T_(reg)17 cells. Insome embodiments, the immune cell is a natural killer (NK) cell. In someembodiments, the immune cell is a B cell. In some embodiments, theimmune cell is a macrophage.

Similarly, the type of second cell depends on the disorder underconsideration. In some embodiments, the second cell (the cell thatexpresses a PD-1 ligand) is a second immune cell, e.g., a regulatoryimmune cell. In some embodiments, the regulatory immune cell is any oneor more of a regulatory T cell, a B cell, a macrophage, amyeloid-derived suppressor cell, a dendritic cell, or a mesenchymalstromal cell. In some embodiments, the regulatory immune cell is aregulatory T cell, e.g., CD8+ T cell or CD4+ T cell.

In some embodiments, the second cell is a tumor cell. As used herein,“tumor cell” is sometimes used interchangeably with “cancer cell”, butalso encompasses non-malignant (non-cancerous) cells exhibitingincreased proliferation as compared to a normal cell. In someembodiments, the tumor cell is a cancer that can be treated by blockingthe interaction between PD-1 expressed by an immune cell and its ligand(e.g., PD-L1 or PD-L2) expressed on a second cell, while bridging theimmune cell and the tumor cell. In some embodiments, the tumor cell isselected from the group consisting of a hematological cancer, alymphoma, a myeloma, a leukemia, a neurological cancer, melanoma, breastcancer, a prostate cancer, a colorectal cancer, lung cancer, head andneck cancer, a gastrointestinal cancer, liver cancer, pancreatic cancer,a genitourinary cancer, a bone cancer, renal cancer, and a vascularcancer. In some embodiments, the tumor cell is selected from the groupconsisting of Kaposi's sarcoma, leukemia, acute lymphocytic leukemia,acute myelocytic leukemia, myeloblasts promyelocyte myelomonocyticmonocytic erythroleukemia, chronic leukemia, chronic myelocytic(granulocytic) leukemia, chronic lymphocytic leukemia, mantle celllymphoma, primary central nervous system lymphoma, Burkitt's lymphomaand marginal zone B cell lymphoma, Polycythemia vera Lymphoma, Hodgkin'sdisease, non-Hodgkin's disease, multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, solid tumors, sarcomas, andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma,osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, uterine cancer, testicular tumor, lung carcinoma, small celllung carcinoma, non-small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basalcell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brainand central nervous system (CNS) cancer, cervical cancer,choriocarcinoma, colorectal cancers, connective tissue cancer, cancer ofthe digestive system, endometrial cancer, esophageal cancer, eye cancer,head and neck cancer, gastric cancer, intraepithelial neoplasm, kidneycancer, larynx cancer, liver cancer, lung cancer (small cell, largecell), melanoma, neuroblastoma; oral cavity cancer (for example lip,tongue, mouth and pharynx), ovarian cancer, pancreatic cancer,retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of therespiratory system, sarcoma, skin cancer, stomach cancer, testicularcancer, thyroid cancer, uterine cancer, and cancer of the urinarysystem.

As described herein, multispecific antigen-binding constructs of thepresent invention include bispecific, tri specific, tetraspecific, ormultispecific antibodies (immunoglobulins) or antigen-binding portionsor fragments thereof.

The term “immunoglobulin” refers to a class of structurally relatedproteins generally comprising two pairs of polypeptide chains: one pairof light (L) chains and one pair of heavy (H) chains. In an “intactimmunoglobulin,” all four of these chains are interconnected bydisulfide bonds. The structure of immunoglobulins has been wellcharacterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5(2013) Lippincott Williams & Wilkins, Philadelphia, Pa. Briefly, eachheavy chain typically comprises a heavy chain variable region (V_(H))and a heavy chain constant region (C_(H)). The heavy chain constantregion typically comprises three domains, C_(H1), C_(H2), and C_(H3).Each light chain typically comprises a light chain variable region(V_(L)) and a light chain constant region. The light chain constantregion typically comprises one domain, abbreviated C_(L). The term“immunoglobulin” (Ig) is sometimes used interchangeably with the term“antibody” herein.

The term “antibody” describes a type of immunoglobulin molecule and isused herein in its broadest sense. An antibody specifically includesintact antibodies (e.g., intact immunoglobulins), and antibody fragmentssuch as antigen-binding fragments of an antibody, as described herein.Thus, “antibody” can refer to an intact antibody as well as anantigen-binding fragment thereof. Antibodies comprise at least oneantigen-binding domain. One example of an antigen-binding domain is anantigen binding domain formed by a V_(H)-V_(L) dimer. Antibodies can bedescribed by the antigen to which they specifically bind. For example, aPD-1 antibody, alternatively referred to as an anti-PD-1 antibody, is anantibody that specifically binds to the inhibitory receptor PD-1.

The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability (“hypervariable regions (HVRs);” also called“complementarity determining regions” (CDRs)) interspersed with regionsthat are more conserved. The more conserved regions are called frameworkregions (FRs). Each V_(H) and V_(L) generally comprises three CDRs andfour FRs, arranged in the following order (from N-terminus toC-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved inantigen binding, and confer antigen specificity and binding affinity tothe antibody. See Kabat et al., Sequences of Proteins of ImmunologicalInterest 5th ed. (1991) Public Health Service, National Institutes ofHealth, Bethesda, Md., incorporated by reference in its entirety.

The light chain from vertebrate species can be assigned to one of twotypes, called kappa and lambda, based on the sequence of the constantdomain.

The heavy chain from vertebrate species can be assigned to one of fivedifferent classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. Theseclasses are also designated α, δ, ε, γ, and μ, respectively. The IgG andIgA classes are further divided into subclasses on the basis ofdifferences in sequence and function. Humans express the followingsubclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

Methods of generating and screening for an antibody against a desiredtarget is well-known in the art. Methods of further modifying antibodiesfor enhanced properties (e.g., enhanced affinity, chimerization,humanization) as well as generating antigen-binding fragments, asdescribed herein, are also well-known in the art.

The term “chimeric antibody” refers to an antibody in which a componentof the heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

“Humanized” forms of non-human antibodies are chimeric antibodies thatcontain minimal sequence derived from the non-human antibody. Ahumanized antibody is generally a human immunoglobulin (recipientantibody) in which residues from one or more CDRs are replaced byresidues from one or more CDRs of a non-human antibody (donor antibody).The donor antibody can be any suitable non-human antibody, such as amouse, rat, rabbit, chicken, or non-human primate antibody having adesired specificity, affinity, or biological effect. In some instances,selected framework region residues of the recipient antibody arereplaced by the corresponding framework region residues from the donorantibody. Humanized antibodies can also comprise residues that are notfound in either the recipient antibody or the donor antibody. Suchmodifications can be made to further refine antibody function. Forfurther details, see Jones et al., (1986) Nature, 321:522-525; Riechmannet al., (1988) Nature, 332:323-329; and Presta, (1992) Curr. Op. Struct.Biol., 2:593-596, each of which is incorporated by reference in itsentirety.

A “human antibody” is one which possesses an amino acid sequencecorresponding to that of an antibody produced by a human or a humancell, or derived from a non-human source that utilizes a human antibodyrepertoire or human antibody-encoding sequences (e.g., obtained fromhuman sources or designed de novo). Human antibodies specificallyexclude humanized antibodies.

In some embodiments, an antibody molecule comprises a diabody, and asingle-chain molecule, as well as an antigen-binding fragment of anantibody (e.g., Fab, F(ab′)₂, and Fv). For example, an antibody moleculecan include a heavy (H) chain variable domain sequence (abbreviatedherein as VH), and a light (L) chain variable domain sequence(abbreviated herein as VL). In some embodiments, an antibody moleculecomprises or consists of a heavy chain and a light chain (referred to asa half antibody). In another example, an antibody molecule includes twoheavy (H) chain variable domain sequences and two light (L) chainvariable domain sequence, thereby forming two antigen binding sites,such as Fab, Fab′, F(ab′)₂, Fc, Fd, Fd′, Fv, single chain antibodies(scFv, for example), single variable domain antibodies, diabodies (Dab)(bivalent and bispecific), and chimeric (e.g., humanized) antibodies,which may be produced by the modification of whole antibodies or thosesynthesized de novo using recombinant DNA technologies. These functionalantibody fragments retain the ability to selectively bind with theirrespective antigen. Antibodies and antibody fragments can be from anyclass of antibodies including, but not limited to, IgG, IgA, IgM, IgD,and IgE, and from any subclass (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, andIgG4) of antibodies. The preparation of antibody molecules can bemonoclonal or polyclonal. An antibody molecule can also be a human,humanized, CDR-grafted, or in vitro generated antibody. The antibody canhave a heavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3,or IgG4. The antibody can also have a light chain chosen from, e.g.,kappa or lambda. In some embodiments, the antibody comprises an IgG1heavy chain constant region having an amino acid sequence that is atleast 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 64(ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK). In some embodiments, the antibodycomprises an IgG4 heavy chain constant region having an amino acidsequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 68(ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK). In some embodiments, the antibodycomprises an IgG4 heavy chain constant region having an amino acidsequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 69(ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG)

Antigen-binding portions or fragments of an antibody molecule are wellknown in the art, and include, for example: (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a diabody(dAb) fragment, which consists of a VH domain; (vi) a camelid orcamelized variable domain; (vii) a single chain Fv (scFv), see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody.These antibody fragments are obtained using conventional techniquesknown to those skilled in the art, and the fragments are screened forutility in the same manner as are intact antibodies.

Antibody molecules can also be single domain antibodies. Single domainantibodies can include antibodies whose complementary determiningregions are part of a single domain polypeptide. Examples include, butare not limited to, heavy chain antibodies, antibodies naturally devoidof light chains, single domain antibodies derived from conventional4-chain antibodies, engineered antibodies and single domain scaffoldsother than those derived from antibodies. Single domain antibodies maybe any of the art, or any future single domain antibodies. Single domainantibodies may be derived from any species including, but not limited tomouse, human, camel, llama, fish, shark, goat, rabbit, and bovine.According to another aspect of the invention, a single domain antibodyis a naturally-occurring single-domain antibody known as heavy chainantibody devoid of light chains. Such single domain antibodies aredisclosed in WO 9404678, for example. For clarity reasons, this variabledomain derived from a heavy chain antibody naturally devoid of lightchain is known herein as a VHH or nanobody to distinguish it from theconventional VH of four chain immunoglobulins. Such a VHH molecule canbe derived from antibodies raised in Camelidae species, for example incamel, llama, dromedary, alpaca and guanaco. Other species besidesCamelidae may produce heavy chain antibodies naturally devoid of lightchain; such VHHs are within the scope of the invention.

In some embodiments, the multispecific antigen-binding constructcomprises a bispecific antibody. A bispecific antibody has specificityfor no more than two antigens, but can have more than two binding sites,as described herein. A bispecific antibody molecule is characterized bya first immunoglobulin variable domain sequence which has bindingspecificity for a first antigen (e.g., PD-1) and a second immunoglobulinvariable domain sequence that has binding specificity for a secondantigen (e.g., a PD-1 ligand such as PD-L1 ligand). In some embodimentsa bispecific antibody molecule comprises a scFv, or fragment thereof,have binding specificity for a first antigen and a scFv, or fragmentthereof, have binding specificity for a second antigen. See, e.g.,Kontermann & Brinkmann, (2015), Drug Discovery Today, 20(7):838-47,incorporated by reference in its entirety.

Various formats and methods are known in the art that can be used togenerate the multivalent and/or multispecific constructs describedherein, such as multivalent and/or multispecific antibody formats ofboth asymmetric and symmetric architectures. Non-limiting examples ofsuch formats include (i) Fc-less bispecific antibody formats, such astandem single-chain variable fragments (scFv2, taFv) and triplebodies,including bi-specific T cell engager (BiTE) and bispecific killer cellengagers (BiKE) molecules; bispecific single-domain antibody fusionproteins comprising single-domain antibodies, such as VH or VL domains,VHH, VNAR and Nanobodies; diabodies and diabody derivatives, includingtandem diabody and dual-affinity retargeting (DART) proteins; Fab fusionproteins; and other Fc-less fusion proteins, through the use ofheterodimerizing peptides or miniantibodies from various proteins, e.g.,leucine zippers with a coiled coil structure; (ii) bispecific IgGs withasymmetric architecture, such as asymmetric IgGs with heavy and lightchains from two different antibodies; bispecific IgGs with an asymmetricFc region using knobs-into-holes approaches, electrostatic interactions(steering) to avoid homodimerization of CH3 domains, preferential heavychain heterodimerization by introducing charge pairs into the hingeregion of IgG1 and IgG2, strand-exchange engineered domain (SEED)heterodimers, and bispecific engagement by antibodies based on the Tcell receptor (BEAT) technologies; asymmetric Fc and CH3 fusionproteins; (iii) bispecific antibodies with a symmetric architecture,such as appended IgGs by fusion of scFvs, fusion of domain antibodiesand scaffold proteins, fusion of Fab arms, and fusion of additionalvariable heavy and light chain domains; modified IgG molecules;symmetric Fc- and CH3-based bispecific antibodies; and bispecificantibodies using immunoglobulin-derived homodimerization domains. See,for example, “The making of bispecific antibodies,” Brinkmann andKontermann, MABS 2017, Vol. 9:2, pp. 182-212, the contents of which areherein incorporated by reference in its entirety. See also, the“knob ina hole” approach described in, e.g., U.S. Pat. No. 5,731,168; theelectrostatic steering Fc pairing as described in, e.g., WO 09/089004,WO 06/106905 and WO 2010/129304; Strand Exchange Engineered Domains(SEED) heterodimer formation as described in, e.g., WO 07/110205; Fabarm exchange as described in, e.g., WO 08/119353, WO 2011/131746, and WO2013/060867; double antibody conjugate, e.g., by antibody cross-linkingto generate a bi-specific structure using a heterobifunctional reagenthaving an amine-reactive group and a sulfhydryl reactive group asdescribed in, e.g., U.S. Pat. No. 4,433,059; bispecific antibodydeterminants generated by recombining half antibodies (heavy-light chainpairs or Fabs) from different antibodies through cycle of reduction andoxidation of disulfide bonds between the two heavy chains, as describedin, e.g., U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., threeFab′ fragments cross-linked through sulfhdryl reactive groups, asdescribed in, e.g., U.S. Pat. No. 5,273,743; biosynthetic bindingproteins, e.g., pair of scFvs cross-linked through C-terminal tailspreferably through disulfide or amine-reactive chemical cross-linking,as described in, e.g., U.S. Pat. No. 5,534,254; bifunctional antibodies,e.g., Fab fragments with different binding specificities dimerizedthrough leucine zippers (e.g., c-fos and c-jun) that have replaced theconstant domain, as described in, e.g., U.S. Pat. No. 5,582,996;bispecific and oligospecific mono- and oligovalent receptors, e.g.,VH-CH1 regions of two antibodies (two Fab fragments) linked through apolypeptide spacer between the CH1 region of one antibody and the VHregion of the other antibody typically with associated light chains, asdescribed in, e.g., U.S. Pat. No. 5,591,828; bispecific DNA-antibodyconjugates, e.g., crosslinking of antibodies or Fab fragments through adouble stranded piece of DNA, as described in, e.g., U.S. Pat. No.5,635,602; bispecific fusion proteins, e.g., an expression constructcontaining two scFvs with a hydrophilic helical peptide linker betweenthem and a full constant region, as described in, e.g., U.S. Pat. No.5,637,481; multivalent and multispecific binding proteins, e.g., dimerof polypeptides having first domain with binding region of Ig heavychain variable region, and second domain with binding region of Ig lightchain variable region, generally termed diabodies (higher orderstructures are also encompassed creating for bispecifc, trispecific, ortetraspecific molecules, as described in, e.g., U.S. Pat. No. 5,837,242;minibody constructs with linked VL and VH chains further connected withpeptide spacers to an antibody hinge region and CH3 region, which can bedimerized to form bispecific/multivalent molecules, as described in,e.g., U.S. Pat. No. 5,837,821; VH and VL domains linked with a shortpeptide linker (e.g., 5 or 10 amino acids) or no linker at all in eitherorientation, which can form dimers to form bispecific diabodies; trimersand tetramers, as described in, e.g., U.S. Pat. No. 5,844,094; String ofVH domains (or VL domains in family members) connected by peptidelinkages with crosslinkable groups at the C-terminus further associatedwith VL domains to form a series of FVs (or scFvs), as described in,e.g., U.S. Pat. No. 5,864,019; and single chain binding polypeptideswith both a VH and a VL domain linked through a peptide linker arecombined into multivalent structures through non-covalent or chemicalcrosslinking to form, e.g., homobivalent, heterobivalent, trivalent, andtetravalent structures using both scFV or diabody type format, asdescribed in, e.g., U.S. Pat. No. 5,869,620. Additional exemplarymultispecific and bispecific molecules and methods of making the sameare found, for example, in U.S. Pat. Nos. 5,910,573, 5,932,448,5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396,6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,185, 6,833,441,7,129,330, 7,183,076, 7,521,056, 7,527,787, 7,534,866, 7,612,181,US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1,US2003211078A1, US2004219643A1, US2004220388A1, US2004242847A1,US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1,US2005100543A1, US2005136049A1, US2005136051A1, US2005163782A1,US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1,US2006263367A1, US2007004909A1, US2007087381A1, US2007128150A1,US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1,US2008069820A1, US2008152645A1, US2008171855A1, US2008241884A1,US2008254512A1, US2008260738A1, US2009130106A1, US2009148905A1,US2009155275A1, US2009162359A1, US2009162360A1, US2009175851A1,US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1,US2009274649A1, EP346087A2, WO0006605A2, WO02072635A2, WO04081051A1,WO06020258A2, WO2007044887A2, WO2007095338A2, WO2007137760A2,WO2008119353A1, WO2009021754A2, WO2009068630A1, WO9103493A1,WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1, WO9637621A2,WO9964460A1. The contents of the above-referenced applications areincorporated herein by reference in their entireties.

In some embodiments, the multispecific antigen-binding construct of thepresent invention is a bispecific antibody. Bispecific antibodiesaccording to the present disclosure can be generated against PD-1 andPD-L1, or against PD-1 and PD-L2. The antibody arms of the bispecificantibody can be generated by standard techniques, as disclosed herein.In some embodiments, any known antibodies against PD-1 and its ligandcan be used to generate a bispecific antibody according to the presentdisclosure. For example, such bispecific constructs have beenexemplified herein (see, for example, Pembrolizumab (PD-1 antibody)joined with Atezolizumab (PD-L1 antibody) in FIG. 3; Nivolumab (PD-1antibody) joined with Atezolizumab (PD-L1 antibody) in FIG. 4). Asexemplified herein, multispecific antigen-binding constructs (e.g.,bispecific antibodies) described herein can be generated using knownand/or available antibodies in the art.

In some embodiments, the bispecific antibody is bivalent—e.g., one armis monovalent for PD-1 while the other arm is monovalent for eitherPD-L1 or PD-L2, or both—e.g., cross-reacts with both ligands). In someembodiments, the bispecific antibody is tetravalent, such as the novelBispecific 3 and Bispecific 4 antibodies described herein. For example,as illustrated in FIG. 3, the Pembrolizumab binding arm is bivalent forPD-1, each binding the same epitope on PD-1, while the Atezolizumabbinding arm is bivalent for PD-L1, each binding the same epitope onPD-L1. This can also be seen in, for example, the bispecific format inFIG. 8 (the exemplary common light chain bispecific illustrated in theleft panel of step 2 of the workflow presented in FIG. 8). The exemplarycommon light chain bispecific format in FIG. 8 (the format illustratedin the right panel of step 2 of the workflow) represents another exampleof a tetravalent bispecific format. In contrast to the tetravalentbispecific format in which the first antigen binding arms are joined tothe second antigen binding arms on opposite ends of the Fc region, hereeach Fab of the first antigen binding arms is joined to each Fab of thesecond antigen binding arm. For example, one a Fab of the first antigenbinding arm is linked to a Fab of the second antigen binding arm using alinker, where each antigen binding arm shares a common light chain. See,FIG. 8, the format illustrated in the right panel of step 2 of theworkflow.

In some embodiments, the bispecific antibody is tetravalent, wherein onearm is bivalent for PD-1, each binding two different epitopes on PD-1.In some embodiments, the bispecific antibody is tetravalent, wherein onearm is bivalent for a ligand of PD-1 (PD-L1 and/or PD-L2), each bindingtwo different epitopes on a ligand of PD-1. In some embodiments, thebispecific antibody is tetravalent, wherein one arm is bivalent forPD-1, each binding two different but overlapping epitopes on PD-1. Insome embodiments, the bispecific antibody is tetravalent, wherein onearm is bivalent for PD-1 ligand (PD-L1 and/or PD-L2), each binding twodifferent but overlapping epitopes on the PD-1 ligand (PD-L1 and/orPD-L2). In some embodiments, the bispecific antibody is tetravalent,wherein one arm is bivalent for PD-1 and each binds the same epitope onPD-1. In some embodiments, the bispecific antibody is tetravalent,wherein one arm is bivalent for a PD-1 ligand (PD-L1 and/or PD-L2), andeach binds the same epitope on the PD-1 ligand (PD-L1 and/or PD-L2). Insome embodiments, the bispecific antibody is tetravalent, where one armis bivalent for a same epitope on PD-1; and the other arm is bivalentfor a same epitope on a PD-1 ligand (PD-L1 and/or PD-L2).

In some embodiments, the bispecific antibody is an antagonist of bothPD-1 and PD-L1. In some embodiments, the bispecific antibody is anantagonist of both PD-1 and PD-L2. In some embodiments, the bispecificantibody is an antagonist of PD-1 and both ligands PD/L1 and PD-L2(e.g., cross reacts with both ligands).

In certain embodiments, the first antigen-binding arm and the secondantigen-binding arm are linked by at least one amino linker amino acidsequence. Optionally, the linker amino acid sequence comprises GGGGSX(SEQ ID NO: 121), wherein x is an integer between and including 1 to 6.

In some embodiments, the multispecific antigen-binding construct doesnot comprise an immunoglobulin Fc domain.

In some embodiments, the construct comprises an immunoglobulin Fcdomain. In some embodiments, the first arm or second arm, or both, ofthe construct, comprises a heavy chain comprising one or moreimmunoglobulin Fc modifications. In some embodiments, the immunoglobulinFc domain of the heavy chain comprises one or more amino acid mutationsthat, e.g., promote heterodimerization of the first and second arms,promote serum half-life, and/or modify effector function. In someembodiments, the mutation is present in a CH3 domain of the heavy chain.See, e.g., Xu et al., mAbs 7(1): 231-42, 2015.

While traditional Fc fusion proteins and antibodies are examples ofunguided interaction pairs, a variety of engineered Fc domains have beendesigned as asymmetric interaction pairs (Spiess et al., (2015)Molecular Immunology 67(2A): 95-106) to promote heterodimerization,e.g., of a first antigen-binding arm and a second antigen-bind arm.Various methods are known in the art that increase desired pairing ofFc-containing polypeptide chains in a single cell line to produce apreferred asymmetric fusion protein at acceptable yields [see, forexample, Klein et al. (2012) mAbs 4:653-663; and Spiess et al. (2015)Molecular Immunology 67(2PartA): 95-106. Methods to obtain desiredpairing of Fc-containing polypeptides include, but are not limited to,charge-based pairing (electrostatic steering), “knobs-into-holes” stericpairing, SEEDbody pairing, and leucine zipper-based pairing. See, forexample, Ridgway et al. (1996) Protein Eng 9:617-621; Merchant et al.(1998) Nat Biotech 16:677-681; Davis et al. (2010) Protein Eng Des Sel23:195-202; Gunasekaran et al. (2010); 285:19637-19646; Wranik et al.(2012) J Biol Chem 287:43331-43339; U.S. Pat. No. 5,932,448; WO1993/011162; WO 2009/089004, and WO 2011/034605.

For example, one means by which interaction between specificpolypeptides may be promoted is by engineering protuberance-into-cavity(knob-into-holes) complementary regions such as described in Arathoon etal., U.S. Pat. No. 7,183,076; Carter et al., U.S. Pat. No. 5,731,168;and Kumar et al., WO 2016/164089, incorporated herein by reference intheir entireties. “Protuberances” are constructed by replacing smallamino acid side chains from the interface of the first polypeptide(e.g., a first interaction pair) with larger side chains (e.g., tyrosineor tryptophan). Complementary “cavities” of identical or similar size tothe protuberances are optionally created on the interface of the secondpolypeptide (e.g., a second interaction pair) by replacing large aminoacid side chains with smaller ones (e.g., alanine or threonine). Where asuitably positioned and dimensioned protuberance or cavity exists at theinterface of either the first or second polypeptide, it is onlynecessary to engineer a corresponding cavity or protuberance,respectively, at the adjacent interface.

At neutral pH (7.0), aspartic acid and glutamic acid are negativelycharged and lysine, arginine, and histidine are positively charged.These charged residues can be used to promote heterodimer formation andat the same time hinder homodimer formation. Attractive interactionstake place between opposite charges and repulsive interactions occurbetween like charges. In part, protein complexes disclosed herein makeuse of the attractive interactions for promoting heteromultimerformation (e.g., heterodimer formation), and optionally repulsiveinteractions for hindering homodimer formation (e.g., homodimerformation) by carrying out site directed mutagenesis of chargedinterface residues.

For example, the IgG1 CH3 domain interface comprises four unique chargeresidue pairs involved in domain-domain interactions: Asp356-Lys439′,Glu357-Lys370′, Lys392-Asp399′, and Asp399-Lys409′ [residue numbering inthe second chain is indicated by (′)]. It should be noted that thenumbering scheme used here to designate residues in the IgG1 CH3 domainconforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetrypresent in the CH3-CH3 domain interactions, each unique interaction willbe represented twice in the structure (e.g., Asp-399-Lys409′ andLys409-Asp399′). In the wild-type sequence, K409-D399′ favors bothheterodimer and homodimer formation. A single mutation switching thecharge polarity (e.g., K409E; positive to negative charge) in the firstchain leads to unfavorable interactions for the formation of the firstchain homodimer. The unfavorable interactions arise due to the repulsiveinteractions occurring between the same charges (negative-negative;K409E-D399′ and D399-K409E′). A similar mutation switching the chargepolarity (D399K′; negative to positive) in the second chain leads tounfavorable interactions (K409′-D399K′ and D399K-K409′) for the secondchain homodimer formation. But, at the same time, these two mutations(K409E and D399K′) lead to favorable interactions (K409E-D399K′ andD399-K409′) for the heterodimer formation. The electrostatic steeringeffect on heterodimer formation and homodimer discouragement can befurther enhanced by mutation of additional charge residues which may ormay not be paired with an oppositely charged residue in the second chainincluding, for example, Arg355 and Lys360. See, e.g., WO 2016/164089.

Thus, in some embodiments, the multispecific antigen-binding constructs(e.g., bispecific constructs) described herein can comprise a constantdomain of an immunoglobulin, including, for example, the Fc portion ofan immunoglobulin. For example, a first arm may comprise an amino acidsequence that is derived from an Fc domain of an IgG (IgG1, IgG2, IgG3,or IgG4), IgA (IgA1 or IgA2), IgE, or IgM immunoglobulin. Optionally, asecond arm may comprise an amino acid sequence that is derived from anFc domain of an IgG (IgG1, lgG2, lgG3, or IgG4), IgA (IgA1 or IgA2),IgE, or IgM. Such immunoglobulin domains may comprise one or more aminoacid modifications (e.g., deletions, additions, and/or substitutions)that promote heterodimer formation. In some embodiments, a multispecificantigen-binding construct is of the IgG1 isotype. In some embodiments, amultispecific antigen-binding construct is of the IgG1 isotype andcomprises a substitution. In some embodiments, a multispecificantigen-binding constructis of the IgG2 isotype. In some embodiments, amultispecific antigen-binding construct is of the IgG3 isotype. In someembodiments, a multispecific antigen-binding construct is of the IgG4isotype. In some embodiments, a multispecific antigen-binding constructis of the IgG4 isotype and comprises a substitution. In someembodiments, the substitution is at Ser228 when numbered according to EUnumbering. In some embodiments, the substitution at Ser228 is S228P. Insome embodiments, a first arm and a second arm comprise Fc domainsderived from the same immunoglobulin class and subtype. In someembodiments, a first arm and a second arm comprise Fc domains derivedfrom different immunoglobulin classes or subtypes. Similarly, a firstarm and/or a second arm (e.g., an asymmetric pair or an unguidedinteraction pair) comprise a modified constant domain of animmunoglobulin, including, for example, one or more amino acidmodifications (e.g., deletions, additions, and/or substitutions) thatpromote heterodimer formation. Methods of generating Fe modificationshaving the desired heterodimer formation are known in the art.

In some embodiments, the Fc domain can be modified to enhance serumhalf-life of the multispecific antigen-binding construct disclosedherein. Fc domains comprising one or more mutations which enhance ordiminish antibody binding to the Fc receptor, e.g., at acidic pH ascompared to neutral pH, are known in the art. For example, theconstructs disclosed herein may comprise a mutation in the C_(H)2 or aC_(H)3 region of the Fc domain, wherein the mutation(s) increases theaffinity of the Fc domain to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0). Such mutationsmay result in an increase in serum half-life of the construct whenadministered to an animal. Methods of modifying the Fc domain fordesired characteristics, such as enhanced serum half-life are known inthe art.

In some embodiments, the constructs described herein comprise an alteredheavy chain constant region that has reduced (or no) effector functionrelative to its corresponding unaltered constant region. Effectorfunctions involving the constant region of the constructs describedherein may be modulated by altering properties of the constant or Fcregion. Altered effector functions include, for example, a modulation inone or more of the following activities: antibody-dependent cellularcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis,binding to one or more Fc-receptors, and pro-inflammatory responses.Modulation refers to an increase, decrease, or elimination of aneffector function activity exhibited by a subject antibody containing analtered constant region as compared to the activity of the unalteredform of the constant region. In particular embodiments, modulationincludes situations in which an activity is abolished or completelyabsent.

An altered constant region with altered FcR binding affinity and/or ADCCactivity and/or altered CDC activity is a polypeptide which has eitheran enhanced or diminished FcR binding activity and/or ADCC activityand/or CDC activity compared to the unaltered form of the constantregion. An altered constant region which displays increased binding toan FcR binds at least one FcR with greater affinity than the unalteredpolypeptide. An altered constant region which displays decreased bindingto an FcR binds at least one FcR with lower affinity than the unalteredform of the constant region. Such variants which display decreasedbinding to an FcR may possess little or no appreciable binding to anFcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42,41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1%) of the binding to the FcR as compared to the level ofbinding of a native sequence immunoglobulin constant or Fc region to theFcR. Similarly, an altered constant region that displays modulated ADCCand/or CDC activity may exhibit either increased or reduced ADCC and/orCDC activity compared to the unaltered constant region. For example, insome embodiments, any one or more of the antibodies described hereincomprising an altered constant region can exhibit approximately 0 to 50%(e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37,36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) ofthe ADCC and/or CDC activity of the unaltered form of the constantregion. A multispecific antigen-binding construct described hereincomprising an altered constant region displaying reduced ADCC and/or CDCcan exhibit reduced or no ADCC and/or CDC activity.

In some embodiments, the multispecific antigen-binding constructsdescribed herein exhibit reduced or no effector function. In someembodiments, the multispecific antigen-binding constructs comprise ahybrid constant region, or a portion thereof, such as a G2/G4 hybridconstant region (see e.g., Burton et al. (1992) Adv Immun 51:1-18;Canfield et al. (1991) J Exp Med 173:1483-1491; and Mueller et al.(1997) Mol Immunol 34(6):441-452).

In some embodiments, the multispecific antigen-binding constructsdescribed herein can contain an altered constant region exhibitingenhanced or reduced complement dependent cytotoxicity (CDC). ModulatedCDC activity can be achieved by introducing one or more amino acidsubstitutions, insertions, or deletions in an Fc region of the antibody.See, e.g., U.S. Pat. No. 6,194,551.

The constructs and antigen-binding arms described herein can comprise,in part, scaffold domains, proteins, or portions, e.g., molecules whichdo not provide target receptor-binding activity, but which can provide aportion or domain of the construct which provides spatial organization,structural support, a means of linking of multiple receptor-bindingunits, or other desired characteristics, e.g., improved half-life.Various scaffold technologies and compositions are known in the art andcan be readily linked or conjugated to the antigen-binding unitsdescribed herein. The scaffold domain, protein, or portion can bederived from an antibody or not derived from an antibody. Such scaffoldproteins, and domains thereof, are, generally, obtained throughcombinatorial chemistry-based adaptation of preexisting antigen-bindingproteins.

Non-antibody protein scaffolds can be considered to fall into twostructural categories, domain-sized constructs (in the range of 6 to 20kDa), and constrained peptides (in the 2-4 kDa range). Domain-sizednon-antibody scaffolds include, but are not limited to, affibodies,affilins, anticalins, atrimers, DARPins, FN3 scaffolds (such asadnectins and centyrins), fynomers, Kunitz domains, pronectins andOBodies. Peptide-sized non-antibody scaffolds include, for example,avimers, bicyclic peptides and cysteine knots. These non-antibodyscaffolds and the underlying proteins or peptides on which they arebased or from which they have been derived are reviewed by, e.g., Simeonand Chen, Protein Cell 9(1): 3-14 (2018); Vazquez-Lombardi et al., DrugDiscovery Today 20: 1271-1283 (2015), and by Binz et al., NatureBiotechnol. 23: 1257-1268 (2005), the contents of each of which areherein incorporated by reference in their entireties. Advantages ofusing non-antibody scaffolds include increased affinity, targetneutralization, and stability. Various non-antibody scaffolds also canovercome some of the limitations of antibody scaffolds, e.g., in termsof tissue penetration, smaller size, and thermostability. Somenon-antibody scaffolds can also permit easier construction, not beinghindered, for example, by the light chain association issue whenbispecific constructs are desired. Methods of constructing constructs ona non-antibody scaffold are known to those of ordinary skill in the art.While not formally on an antibody scaffold, such constructs ofteninclude antibody binding domains, whether in the form of single-domainantibodies, scFvs or other antibody binding-domain variants that providespecific target-binding capabilities.

Accordingly, in some embodiments of any of the aspects described herein,a construct can comprise a non-antibody scaffold protein. In someembodiments of any of the aspects described herein, at least one of thereceptor-binding units can comprise a non-antibody scaffold protein. Oneof skill in the art would appreciate that the scaffold portion of anon-antibody scaffold protein can include, in some embodiments, e.g., anadnectin scaffold or a portion derived from human tenth fibronectin typeIII domain (10Fn3); an anticalin scaffold derived from human lipocalin(e.g., such as those described in, e.g., WO2015/104406); an avimerscaffold or a protein fragment derived from the A-domain of lowdensity-related protein (LRP) and/or very low density lipoproteinreceptor (VLDLR); a fynomer scaffold or portion of the SH3 domain of FYNtyrosine kinase; a kunitz domain scaffold or portion of Kunitz-typeprotease inhibitors, such as a human trypsin inhibitor, aprotinin(bovine pancreatic trypsin inhibitor), Alzheimer's amyloid precursorprotein, and tissue factor pathway inhibitor; a knottin scaffold(cysteine knot miniproteins), such as one based on a trypsin inhibitorfrom E. elaterium; an affibody scaffold or all or part of the Z domainof S. aureus protein A; a β-Hairpin mimetic scaffold; a Designed ankyrinrepeat protein (DARPin) scaffold or artificial protein scaffolds basedon ankyrin repeat (AR) proteins; or any scaffold derived or based onhuman transferrin, human CTLA-4, human crystallin, and human ubiquitin.For example, the binding site of human transferrin for human transferrinreceptor can be diversified to create a diverse library of transferrinvariants, some of which have acquired affinity for different antigens.See, e.g., Ali et al. (1999) J. Biol. Chem. 274:24066-24073. The portionof human transferrin not involved with binding the receptor remainsunchanged and serves as a scaffold, like framework regions ofantibodies, to present the variant binding sites. The libraries are thenscreened, as an antibody library is, and in accordance with the methodsdescribed herein, against a target antigen of interest to identify thosevariants having optimal selectivity and affinity for the target antigen.See, e.g., Hey et al. (2005) TRENDS Biotechnol. 23(10):514-522.

D. Methods for Producing the Multispecific Antigen-Binding Constructs

The disclosure also features methods for producing any of themultispecific antigen-binding constructs described herein. In someembodiments, methods for producing the construct of the presentinvention includes methods for preparing an antibody, and/or fragmentsthereof as described herein. Such methods are well-known in the art, andcan include, e.g., immunizing a subject (e.g., a non-human mammal) withan appropriate immunogen. For example, to generate an antibody thatbinds to PD-1, a skilled artisan can immunize a suitable subject (e.g.,a non-human mammal such as a rat, a mouse, a gerbil, a hamster, a dog, acat, a pig, a goat, a horse, or a non-human primate) with a full-lengthPD-1 polypeptide such as a full-length human PD-1 polypeptide comprisingthe amino acid sequence depicted in SEQ ID NO.: 114 (GenBank accessionnumber NP_005009.2; UniProt Q15116), an antigenic fragment thereof,and/or variant thereof. Similarly, to generate an antibody that binds toa ligand of PD-1 (e.g., PD-L1), a skilled artisan can immunize asuitable subject with a full-length PD-L1 polypeptide such as afull-length human PD-L1 polypeptide comprising the amino acid sequencedepicted in SEQ ID NO.: 115 (GenBank accession number NP 054862.1,UniProt Q9NZQ7), an antigenic fragment thereof, and/or variant thereof.Similarly, to generate an antibody that binds to PD-L2, a skilledartisan can immunize a suitable subject with a full-length PD-L2polypeptide such as a full-length human PD-L2 polypeptide comprising theamino acid sequence depicted in SEQ ID NO.: 116 (GenBank accessionnumber NP_079515.2, UniProt Q9BQ51), an antigenic fragment thereof,and/or variant thereof.

As those of skill in the art would recognize, a full-length polypeptide(PD-1, PD-L1, or PD-L2) can be used as an antigen and antibodies can bescreened for desired binding properties (e.g., blocks PD-1/ligandinteraction; capacity to bridge cells on which PD-1 and its ligand areexpressed). As those of skill in the art would also recognize, antigenicfragments of a polypeptide (PD-1, PD-L1, or PD-L2) can be selected basedon known structural features of the polypeptide. For example, thePD-1/PD-L1 and PD-1/PD-L2 interactions have been structurallywell-characterized (see, e.g., Zak, K., et al. (2015) Structure23(12):2341-48; Ghiotto, M., et al. (2010) Int'l Immuno. 22(8):651-60;Freeman, G. (2008) PNAS 105(30):10275-76; Lazar-Molnar, E. et al. (2008)PNAS 105:10483-88, incorporated by reference in their entireties). Thus,regions within, e.g., PD-1, PD-L1, and/or PD-L2, based onreceptor/ligand interface information available in the art, can be usedto design a suitable antigenic fragment having desired bindingproperties. For example, the PD-1 ectodomain contains a single IgVdomain typical of the CD28 family, wherein PD-L1 and PD-L2 are composedof IgV and IgC domains typical of the B7 family. The structures of PD-1,PD-L1 and/or PD-L2 show a 1:1 stoichiometry, with interaction primarilybetween the faces of the IgV domains. An IgV domain comprises about 120amino acids organized into nine parallel beta strands (ABCC′C″DEFG) withloops connecting the strands. It has been shown that PD-1 uses the frontbeta-face (GFCC′ strands and CC′, CC″, and FG loops) to bind to thebeta-face of PD-L1 (GFCC′) or PD-L2 (AGFC strands and FG loop). Further,six amino acids of the C, F, and G strands of PD-1 form a concave,hydrophobic core that interacts with the F and G strands as well as theFG loop of PD-L2. Eight if 14 residues involved in binding to PD-1 areindentical or highly conserved between PD-L1 and PD-L2. Using suchinformation, those of skill in the art can determine suitable antigenicregions to generate antibodies having desirable properties. For example,those of skill in the art can generate an antibody that cross reactswith both ligands PD-L1 and PD-L2 (see, e.g., U.S. Pat. No. 9,845,356).

A suitable subject (e.g., a non-human mammal) can be immunized with theappropriate antigen along with subsequent booster immunizations a numberof times sufficient to elicit the production of an antibody by themammal. The immunogen can be administered to a subject (e.g., anon-human mammal) with an adjuvant. Adjuvants useful in producing anantibody in a subject include, but are not limited to, proteinadjuvants; bacterial adjuvants, e.g., whole bacteria (BCG,Corynebacterium parvum or Salmonella minnesota) and bacterial componentsincluding cell wall skeleton, trehalose dimycolate, monophosphoryl lipidA, methanol extractable residue (MER) of tubercle bacillus, complete orincomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g.,aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate. Otheradjuvants that can be used in the methods for inducing an immuneresponse include, e.g., cholera toxin and parapoxvirus proteins. Seealso Bieg et al. (1999) Autoimmunity 31(1):15-24. See also, e.g.,Lodmell et al. (2000) Vaccine 18:1059-1066; Johnson et al. (1999) J MedChem 42:4640-4649; Baldridge et al. (1999) Methods 19:103-107; and Guptaet al. (1995) Vaccine 13(14): 1263-1276.

In some embodiments, the methods include preparing a hybridoma cell linethat secretes a monoclonal antibody that binds to the immunogen. Forexample, a suitable mammal such as a laboratory mouse is immunized witha polypeptide (e.g., PD-1, PD-L1, PD-L2) or antigenic fragment asdescribed above. Antibody-producing cells (e.g., B cells of the spleen)of the immunized mammal can be isolated two to four days after at leastone booster immunization of the immunogen and then grown briefly inculture before fusion with cells of a suitable myeloma cell line. Thecells can be fused in the presence of a fusion promoter such as, e.g.,vaccinia virus or polyethylene glycol. The hybrid cells obtained in thefusion are cloned, and cell clones secreting the desired antibodies areselected. For example, spleen cells of Balb/c mice immunized with asuitable immunogen can be fused with cells of the myeloma cell line PAIor the myeloma cell line Sp2/0-Ag 14. After the fusion, the cells areexpanded in suitable culture medium, which is supplemented with aselection medium, for example HAT medium, at regular intervals in orderto prevent normal myeloma cells from overgrowing the desired hybridomacells. The obtained hybridoma cells are then screened for secretion ofthe desired antibodies, e.g., an antibody that binds to PD-1.

In some embodiments, a skilled artisan can identify an anti-PD-1antibody from a non-immune biased library as described in, e.g., U.S.Pat. No. 6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt etal. (2005) Nucleic Acids Res 33(9):e81.

In some embodiments, the methods described herein can involve, or beused in conjunction with, e.g., phage display technologies, bacterialdisplay, yeast surface display, eukaryotic viral display, mammalian celldisplay, and cell-free (e.g., ribosomal display) antibody screeningtechniques (see, e.g., Etz et al. (2001) J Bacteriol 183:6924-6935;Cornelis (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000)Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng10:1303-1310; Yeung et al. (2002) Biotechnol Prog 18:212-220; Boder etal. (2000) Methods Enzymology 328:430-444; Grabherr et al. (2001) CombChem High Throughput Screen 4:185-192; Michael et al. (1995) Gene Ther2:660-668; Pereboev et al. (2001) J Virol 75:7107-7113; Schaffitzel etal. (1999) J Immunol Methods 231:119-135; and Hanes et al. (2000) NatBiotechnol 18:1287-1292).

Methods for identifying antibodies using various phage display methodsare known in the art. In phage display methods, functional antibodydomains are displayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. Such phage can be utilized todisplay antigen-binding domains of antibodies, such as Fab, Fv, ordisulfide-bond stabilized Fv antibody fragments, expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Phage used in these methods are typically filamentous phage such as fdand M13. The antigen binding domains are expressed as arecombinantly-fused protein to any of the phage coat proteins pIII,pVIII, or pIX. See, e.g., Shi et al. (2010) JMB 397:385-396. Examples ofphage display methods that can be used to make the immunoglobulins, orfragments thereof, described herein include those disclosed in Brinkmanet al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995) J ImmunolMethods 184:177-186; Kettleborough et al. (1994) Eur J Immunol24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994)Advances in Immunology 57:191-280; and PCT publication nos. WO 90/02809,WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO95/20401. Suitable methods are also described in, e.g., U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108.

In some embodiments, the phage display antibody libraries can begenerated using mRNA collected from B cells from the immunized mammals.For example, a splenic cell sample comprising B cells can be isolatedfrom mice immunized with a PD-1 polypeptide as described above. mRNA canbe isolated from the cells and converted to cDNA using standardmolecular biology techniques. See, e.g., Sambrook et al. (1989)“Molecular Cloning: A Laboratory Manual, 2^(nd) Edition,” Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane(1988), supra; Benny K. C. Lo (2004), supra; and Borrebaek (1995),supra. The cDNA coding for the variable regions of the heavy chain andlight chain polypeptides of immunoglobulins are used to construct thephage display library. Methods for generating such a library aredescribed in, e.g., Merz et al. (1995) J Neurosci Methods 62(1-2):213-9;Di Niro et al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al.(1995) Methods Mol Biol 51:355-376.

In some embodiments, a combination of selection and screening can beemployed to identify an antibody of interest from, e.g., a population ofhybridoma-derived antibodies or a phage display antibody library.Suitable methods are known in the art and are described in, e.g.,Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al.(1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994),supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. Forexample, a plurality of phagemid vectors, each encoding a fusion proteinof a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of M13 phage)and a different antigen-combining region are produced using standardmolecular biology techniques and then introduced into a population ofbacteria (e.g., E. coli). Expression of the bacteriophage in bacteriacan, in some embodiments, require use of a helper phage. In someembodiments, no helper phage is required (see, e.g., Chasteen et al.,(2006) Nucleic Acids Res 34(21):e145). Phage produced from the bacteriaare recovered and then contacted to, e.g., a target antigen bound to asolid support (immobilized). Phage may also be contacted to antigen insolution, and the complex is subsequently bound to a solid support.

A subpopulation of antibodies screened using the above methods can becharacterized for their specificity and binding affinity for aparticular antigen (e.g., human PD-1) using any immunological orbiochemical based method known in the art. For example, specific bindingof an antibody to PD-1, may be determined for example usingimmunological or biochemical based methods such as, but not limited to,an ELISA assay, SPR assays, immunoprecipitation assay, affinitychromatography, and equilibrium dialysis as described above.Immunoassays which can be used to analyze immunospecific binding andcross-reactivity of the antibodies include, but are not limited to,competitive and non-competitive assay systems using techniques such asWestern blots, RIA, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, immunodiffusionassays, agglutination assays, complement-fixation assays,immunoradiometric assays, fluorescent immunoassays, and protein Aimmunoassays. Such assays are routine and well known in the art.

It is understood that the above methods can also be used to determineif, e.g., an anti-PD-1 antibody does not bind to full-length, human PD-1and/or PD-1 proteins.

In embodiments where the selected CDR amino acid sequences are shortsequences (e.g., fewer than 10-15 amino acids in length), nucleic acidsencoding the CDRs can be chemically synthesized as described in, e.g.,Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1):129-130 andU.S. Pat. No. 6,995,259. For a given nucleic acid sequence encoding anacceptor antibody, the region of the nucleic acid sequence encoding theCDRs can be replaced with the chemically synthesized nucleic acids usingstandard molecular biology techniques. The 5′ and 3′ ends of thechemically synthesized nucleic acids can be synthesized to comprisesticky end restriction enzyme sites for use in cloning the nucleic acidsinto the nucleic acid encoding the variable region of the donorantibody. Alternatively, fragments of chemically synthesized nucleicacids, together capable of encoding an antibody, can be joined togetherusing DNA assembly techniques known in the art (e.g. Gibson Assembly).

Any antibody of choice can be further modified to generate anantigen-binding fragment, as described herein, and/or manipulated usingknown techniques in the art to generate the multispecificantigen-binding constructs as described herein. For example,cross-linking methods can be used to generate a bispecific structureusing a heterobifunctional reagent having an amine-reactive group and asulfhydryl reactive group as described in, e.g., U.S. Pat. No.4,433,059; bispecific antibody determinants can be generated byrecombining half antibodies (heavy-light chain pairs or Fabs) fromdifferent antibodies through cycle of reduction and oxidation ofdisulfide bonds between the two heavy chains, as described in, e.g.,U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., three Fab′fragments can be cross-linked through sulfhdryl reactive groups, asdescribed in, e.g., U.S. Pat. No. 5,273,743. Other methods of generatingbispecific constructs, e.g., methods of generating bispecific constructshaving common light chains, are described herein. Non-limiting examplesof amino acid sequences of common light chains used in the constructsdescribed herein include SEQ ID NOs: 59-63.

E. Expression and Purification of Multispecific Antigen-BindingConstructs

The multispecific antigen-binding constructs thereof described hereincan be produced using a variety of techniques known in the art ofmolecular biology and protein chemistry. For example, a nucleic acidencoding the multispecific antigen-binding construct (as a singlemultifunctional polypeptide, or as separate molecules of a multimericcomplex—e.g., one antigen-binding arm separately from the otherantigen-binding arm) can be inserted into an expression vector thatcontains transcriptional and translational regulatory sequences, whichinclude, e.g., promoter sequences, ribosomal binding sites,transcriptional start and stop sequences, translational start and stopsequences, transcription terminator signals, polyadenylation signals,and enhancer or activator sequences. The regulatory sequences include apromoter and transcriptional start and stop sequences. In addition, theexpression vector can include more than one replication system such thatit can be maintained in two different organisms, for example inmammalian or insect cells for expression and in a prokaryotic host forcloning and amplification.

Several possible vector systems are available for the expression ofcloned heavy chain and light chain polypeptides from nucleic acids inmammalian cells. One class of vectors relies upon the integration of thedesired gene sequences into the host cell genome. Cells which havestably integrated DNA can be selected by simultaneously introducing drugresistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc NatlAcad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol ApplGenet 1:327). The selectable marker gene can be either linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection (Wigler et al. (1979) Cell 16:77). A second class ofvectors utilizes DNA elements which confer autonomously replicatingcapabilities to an extrachromosomal plasmid. These vectors can bederived from animal viruses, such as bovine papillomavirus (Sarver etal. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyomavirus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40virus (Lusky and Botchan (1981) Nature 293:79).

The expression vectors can be introduced into cells in a manner suitablefor subsequent expression of the nucleic acid. The method ofintroduction is largely dictated by the targeted cell type, discussedbelow. Exemplary methods include CaPO₄ precipitation, liposome fusion,cationic liposomes, electroporation, viral infection, dextran-mediatedtransfection, polybrene-mediated transfection, protoplast fusion, anddirect microinjection.

Appropriate host cells for the expression of antibodies orantigen-binding fragments thereof include yeast, bacteria, insect,plant, and mammalian cells. Of particular interest are bacteria such asE. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris,insect cells such as SF9, mammalian cell lines (e.g., human cell lines),as well as primary cell lines.

In some embodiments, an antibody or fragment thereof can be expressedin, and purified from, transgenic animals (e.g., transgenic mammals).For example, an antibody can be produced in transgenic non-human mammals(e.g., rodents) and isolated from milk as described in, e.g., Houdebine(2002) Curr Opin Biotechnol 13(6):625-629; van Kuik-Romeijn et al.(2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J ImmunolMethods 231(1-2):147-157.

The antibodies and fragments thereof can be produced from the cells byculturing a host cell transformed with the expression vector containingnucleic acid encoding the antibodies or fragments, under conditions, andfor an amount of time, sufficient to allow expression of the proteins.Such conditions for protein expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. For example,antibodies expressed in E. coli can be refolded from inclusion bodies(see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expressionsystems and methods for their use are well known in the art (see CurrentProtocols in Molecular Biology, Wiley & Sons, and Molecular Cloning—ALaboratory Manual—3rd Ed., Cold Spring Harbor Laboratory Press, New York(2001)). The choice of codons, suitable expression vectors and suitablehost cells will vary depending on a number of factors, and may be easilyoptimized as needed. An antibody (or fragment thereof) described hereincan be expressed in mammalian cells or in other expression systemsincluding but not limited to yeast, baculovirus, and in vitro expressionsystems (see, e.g., Kaszubska et al. (2000) Protein Expression andPurification 18:213-220).

Following expression, the antibodies and fragments thereof can beisolated. An antibody or fragment thereof can be isolated or purified ina variety of ways known to those skilled in the art depending on whatother components are present in the sample. Standard purificationmethods include electrophoretic, molecular, immunological, andchromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography. For example, anantibody can be purified using a standard anti-antibody column (e.g., aprotein-A or protein-G column). Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.See, e.g., Scopes (1994) “Protein Purification, 3^(rd) edition,”Springer-Verlag, New York City, N.Y. The degree of purificationnecessary will vary depending on the desired use. In some instances, nopurification of the expressed antibody or fragments thereof will benecessary.

Methods for determining the yield or purity of a purified antibody orfragment thereof are known in the art and include, e.g., Bradford assay,UV spectroscopy, Biuret protein assay, Lowry protein assay, amido blackprotein assay, high pressure liquid chromatography (HPLC), massspectrometry (MS), and gel electrophoretic methods (e.g., using aprotein stain such as Coomassie Blue or colloidal silver stain).

F. Modification of the Multispecific Antigen-Binding Constructs

The multispecific antigen-binding constructs can be modified followingtheir expression and purification as a single multifunctionalpolypeptide, or as separate molecules of a multimeric complex—e.g., oneantigen-binding arm separately from the other antigen-binding arm. Themodifications can be covalent or non-covalent modifications. Suchmodifications can be introduced into the antibodies or antigen-bindingfragments by, e.g., reacting targeted amino acid residues of thepolypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. Suitable sitesfor modification can be chosen using any of a variety of criteriaincluding, e.g., structural analysis or amino acid sequence analysis ofthe antibodies or fragments.

The amino acid sequences provided herein are set forth in single-letteramino acid code which can be used interchangeably with three-letteramino acid code. An amino acid refers to any monomer unit that can beincorporated into a peptide, polypeptide, or protein. The twenty naturalor genetically encoded alpha-amino acids are as follows: alanine (Ala orA), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp orD), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E),glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine(Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Pheor F), proline (Pro or P), serine (Ser or S), threonine (Thr or T),tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). Thestructures of these twenty natural amino acids are shown in, e.g.,Stryer et al., Biochemistry, 5^(th)ed., Freeman and Company (2002). Theterm amino acid also includes unnatural amino acids, modified aminoacids (e.g., having modified side chains and/or backbones), and aminoacid analogs.

The terms identical or percent identity, in the context of two or morenucleic acids or polypeptide sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same (e.g., 90%, or 95%or greater identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection.

Identity or similarity with respect to a sequence is defined as thepercentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) with the starting amino acid residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, for example, by the localhomology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970),by the homology alignment algorithm of Needleman and Wunsch (J. Mol.Biol. 48:443, 1970), by the search for similarity method of Pearson andLipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

As with all peptides, polypeptides, and proteins, including fragmentsthereof, it is understood that additional modifications in the aminoacid sequence of the constructs, antibodies or antigen-binding portionsthereof described herein, for example, in the heavy chain variableregion and/or light chain variable region, can occur that do not alterthe nature or function of the antibodies or antigen-binding fragmentsthereof. Such modifications include conservative amino acidssubstitutions, such that each recited sequence optionally contains oneor more conservative amino acid substitutions. The following groups eachcontain amino acids that are conservative substitutions for one another.These groups are exemplary as other conservative substitutions are knownto those of skill in the art.

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

By way of example, when an aspartic acid at a specific residue ismentioned, also contemplated is a conservative substitution at theresidue, for example, glutamic acid. Nonconservative substitutions, forexample, substituting a proline with glycine, are also contemplated.

In some embodiments, the constructs, antibodies or antigen-bindingportions thereof can be conjugated to a heterologous moiety. Theheterologous moiety can be, e.g., a heterologous polypeptide, atherapeutic agent (e.g., a toxin or a drug), or a detectable label suchas, but not limited to, a radioactive label, an enzymatic label, afluorescent label, a heavy metal label, a luminescent label, or anaffinity tag such as biotin or streptavidin. Suitable heterologouspolypeptides include, e.g., an antigenic tag (e.g., FLAG (DYKDDDDK) (SEQID NO: 117), polyhistidine (6-His; HHHHHH (SEQ ID NO: 118)),hemagglutinin (HA; YPYDVPDYA (SEQ ID NO: 119)),glutathione-S-transferase (GST), or maltose-binding protein (MBP)) foruse in purifying the antibodies or fragments. Heterologous polypeptidesalso include polypeptides (e.g., enzymes) that are useful as diagnosticor detectable markers, for example, luciferase, a fluorescent protein(e.g., green fluorescent protein (GFP)), or chloramphenicol acetyltransferase (CAT). Suitable radioactive labels include, e.g., ³²P, ³³P,¹⁴C, ¹²⁵I, ¹³¹I, ³⁵S, and ³H. Suitable fluorescent labels include,without limitation, fluorescein, fluorescein isothiocyanate (FITC),green fluorescent protein (GFP), DYLIGHT™ 488, phycoerythrin (PE),propidium iodide (PI), PerCP, PE-ALEXA FLUOR® 700, Cy5, allophycocyanin,and Cy7. Luminescent labels include, e.g., any of a variety ofluminescent lanthanide (e.g., europium or terbium) chelates. Forexample, suitable europium chelates include the europium chelate ofdiethylene triamine pentaacetic acid (DTPA) ortetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labelsinclude, e.g., alkaline phosphatase, CAT, luciferase, and horseradishperoxidase.

Two proteins (e.g., an antibody and a heterologous moiety) can becross-linked using any of a number of known chemical cross linkers.Examples of such cross linkers are those which link two amino acidresidues via a linkage that includes a “hindered” disulfide bond. Inthese linkages, a disulfide bond within the cross-linking unit isprotected (by hindering groups on either side of the disulfide bond)from reduction by the action, for example, of reduced glutathione or theenzyme disulfide reductase. One suitable reagent,4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio) toluene (SMPT),forms such a linkage between two proteins utilizing a terminal lysine onone of the proteins and a terminal cysteine on the other.Heterobifunctional reagents that cross-link by a different couplingmoiety on each protein can also be used. Other useful cross-linkersinclude, without limitation, reagents which link two amino groups (e.g.,N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g.,1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and acarboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an aminogroup and a guanidinium group that is present in the side chain ofarginine (e.g., p-azidophenyl glyoxal monohydrate).

In some embodiments, a radioactive label can be directly conjugated tothe amino acid backbone of the antibody. Alternatively, the radioactivelabel can be included as part of a larger molecule (e.g., ¹²⁵I inmeta-[¹²⁵I]iodophenyl-N-hydroxysuccinimide ([¹²⁵I]mIPNHS) which binds tofree amino groups to form meta-iodophenyl (mIP) derivatives of relevantproteins (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) orchelate (e.g., to DOTA or DTPA) which is in turn bound to the proteinbackbone. Methods of conjugating the radioactive labels or largermolecules/chelates containing them to the antibodies or antigen-bindingfragments described herein are known in the art. Such methods involveincubating the proteins with the radioactive label under conditions(e.g., pH, salt concentration, and/or temperature) that facilitatebinding of the radioactive label or chelate to the protein (see, e.g.,U.S. Pat. No. 6,001,329).

Methods for conjugating a fluorescent label (sometimes referred to as a“fluorophore”) to a protein (e.g., an antibody) are known in the art ofprotein chemistry. For example, fluorophores can be conjugated to freeamino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines)of proteins using succinimidyl (NETS) ester or tetrafluorophenyl (TFP)ester moieties attached to the fluorophores. In some embodiments, thefluorophores can be conjugated to a heterobifunctional cross-linkermoiety such as sulfo-SMCC. Suitable conjugation methods involveincubating an antibody protein, or fragment thereof, with thefluorophore under conditions that facilitate binding of the fluorophoreto the protein. See, e.g., Welch and Redvanly (2003) “Handbook ofRadiopharmaceuticals: Radiochemistry and Applications,” John Wiley andSons (ISBN 0471495603).

In some embodiments, the antibodies or fragments can be modified, e.g.,with a moiety that improves the stabilization and/or retention of theantibodies in circulation, e.g., in blood, serum, or other tissues. Forexample, the antibody or fragment can be PEGylated as described in,e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al.(2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al.(2002) Advanced Drug Delivery Reviews 54:459-476 or HESylated (FreseniusKabi, Germany; see, e.g., Pavisić et al. (2010) Int J Pharm387(1-2):110-119). The stabilization moiety can improve the stability,or retention of, the antibody (or fragment) by at least 1.5 (e.g., atleast 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

In some embodiments, the antibodies or antigen-binding fragments thereofdescribed herein can be glycosylated. In some embodiments, an antibodyor antigen-binding fragment thereof described herein can be subjected toenzymatic or chemical treatment, or produced from a cell, such that theantibody or fragment has reduced or absent glycosylation. Methods forproducing antibodies with reduced glycosylation are known in the art anddescribed in, e.g., U.S. Pat. No. 6,933,368; Wright et al. (1991) EMBO J10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361. In someembodiments, the antibodies or antigen-binding fragments thereof areaglycosylated.

G. Pharmaceutical Compositions and Formulations

The present disclosure also provides for a pharmaceutical compositioncomprising the multispecific antigen-binding constructs of the presentdisclosure with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant to be used withthe methods disclosed herein. Such pharmaceutical compositions can beused in a subject having e.g., cancer, as disclosed herein.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, the formulation material(s) are for s.c. and/orI.V. administration. In certain embodiments, the pharmaceuticalcomposition can contain formulation materials for modifying, maintainingor preserving, for example, the pH, osmolality, viscosity, clarity,color, isotonicity, odor, sterility, stability, rate of dissolution orrelease, adsorption or penetration of the composition. In certainembodiments, suitable formulation materials include, but are not limitedto, amino acids (such as glycine, glutamine, asparagine, arginine orlysine); antimicrobials; antioxidants (such as ascorbic acid, sodiumsulfite or sodium hydrogen-sulfite); buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates or other organic acids);bulking agents (such as mannitol or glycine); chelating agents (such asethylenediamine tetraacetic acid (EDTA)); complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company (1995). In certain embodiments, the formulationcomprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH5.2, 9% Sucrose. In certain embodiments, the optimal pharmaceuticalcomposition will be determined by one skilled in the art depending upon,for example, the intended route of administration, delivery format anddesired dosage. See, for example, Remington's Pharmaceutical Sciences,supra. In certain embodiments, such compositions may influence thephysical state, stability, rate of in vivo release and/or rate of invivo clearance of the multispecific antigen-binding construct.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Incertain embodiments, the saline comprises isotonic phosphate-bufferedsaline. In certain embodiments, neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In certainembodiments, pharmaceutical compositions comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can furtherinclude sorbitol or a suitable substitute therefore. In certainembodiments, a composition comprising the multispecific antigen-bindingconstructs disclosed herein can be prepared for storage by mixing theselected composition having the desired degree of purity with optionalformulation agents (Remington's Pharmaceutical Sciences, supra) in theform of a lyophilized cake or an aqueous solution. Further, in certainembodiments, a composition comprising the multispecific antigen-bindingconstruct disclosed herein can be formulated as a lyophilizate usingappropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a multispecificantigen-binding construct, in a pharmaceutically acceptable vehicle. Incertain embodiments, a vehicle for parenteral injection is steriledistilled water in which a multispecific antigen-binding construct isformulated as a sterile, isotonic solution, and properly preserved. Incertain embodiments, the preparation can involve the formulation of thedesired molecule with an agent, such as injectable microspheres,bio-erodible particles, polymeric compounds (such as polylactic acid orpolyglycolic acid), beads or liposomes, that can provide for thecontrolled or sustained release of the product which can then bedelivered via a depot injection. In certain embodiments, hyaluronic acidcan also be used, and can have the effect of promoting sustainedduration in the circulation. In certain embodiments, implantable drugdelivery devices can be used to introduce the desired molecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, a multispecific antigen-bindingconstruct can be formulated as a dry powder for inhalation. In certainembodiments, an inhalation solution comprising a multispecificantigen-binding construct can be formulated with a propellant foraerosol delivery. In certain embodiments, solutions can be nebulized.Pulmonary administration is further described in PCT application No.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, a multispecificantigen-binding construct that is administered in this fashion can beformulated with or without those carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Incertain embodiments, a capsule can be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. In certain embodiments, at least one additional agent can beincluded to facilitate absorption of a multispecific antigen-bindingconstruct. In certain embodiments, diluents, flavorings, low meltingpoint waxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of a multispecific antigen-binding construct in amixture with non-toxic excipients which are suitable for the manufactureof tablets. In certain embodiments, by dissolving the tablets in sterilewater, or another appropriate vehicle, solutions can be prepared inunit-dose form. In certain embodiments, suitable excipients include, butare not limited to, inert diluents, such as calcium carbonate, sodiumcarbonate or bicarbonate, lactose, or calcium phosphate; or bindingagents, such as starch, gelatin, or acacia; or lubricating agents suchas magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving a multispecificantigen-binding construct in sustained- or controlled-deliveryformulations. In certain embodiments, techniques for formulating avariety of other sustained- or controlled-delivery means, such asliposome carriers, bio-erodible microparticles or porous beads and depotinjections, are also known to those skilled in the art. See for example,PCT Application No. PCT/US93/00829 which describes the controlledrelease of porous polymeric microparticles for the delivery ofpharmaceutical compositions. In certain embodiments, sustained-releasepreparations can include semipermeable polymer matrices in the form ofshaped articles, e.g. films, or microcapsules. Sustained releasematrices can include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919 and EP 058,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). In certain embodiments, sustained release compositions canalso include liposomes, which can be prepared by any of several methodsknown in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA,82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising a multispecific antigen-binding construct to beemployed therapeutically will depend, for example, upon the therapeuticcontext and objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment, according to certainembodiments, will thus vary depending, in part, upon the moleculedelivered, the indication for which a multispecific antigen-bindingconstruct is being used, the route of administration, and the size (bodyweight, body surface or organ size) and/or condition (the age andgeneral health) of the patient. In certain embodiments, the cliniciancan titer the dosage and modify the route of administration to obtainthe optimal therapeutic effect.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of a multispecific antigen-bindingconstruct in the formulation used. In certain embodiments, a clinicianwill administer the composition until a dosage is reached that achievesthe desired effect. In certain embodiments, the composition cantherefore be administered as a single dose or as two or more doses(which may or may not contain the same amount of the desired molecule)over time, or as a continuous infusion via an implantation device orcatheter. Further refinement of the appropriate dosage is routinely madeby those of ordinary skill in the art and is within the ambit of tasksroutinely performed by them. In certain embodiments, appropriate dosagescan be ascertained through use of appropriate dose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.In certain embodiments, individual elements of the combination therapymay be administered by different routes.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration. In certain embodiments, it can be desirable to use apharmaceutical composition comprising a multispecific antigen-bindingconstruct in an ex vivo manner. In such instances, cells, tissues and/ororgans that have been removed from the patient are exposed to apharmaceutical composition comprising a multispecific antigen-bindingconstruct after which the cells, tissues and/or organs are subsequentlyimplanted back into the patient.

In certain embodiments, a multispecific antigen-binding construct can bedelivered by implanting certain cells that have been geneticallyengineered, using methods such as those described herein, to express andsecrete the polypeptides. In certain embodiments, such cells can beanimal or human cells, and can be autologous, heterologous, orxenogeneic. In certain embodiments, the cells can be immortalized. Incertain embodiments, in order to decrease the chance of an immunologicalresponse, the cells can be encapsulated to avoid infiltration ofsurrounding tissues. In certain embodiments, the encapsulation materialsare typically biocompatible, semi-permeable polymeric enclosures ormembranes that allow the release of the protein product(s) but preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

H. Methods of Use

As described herein, the present disclosure provides a method oftreating a proliferative disorder in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a multispecific antigen-binding construct of the presentdisclosure. In some embodiments, the present disclosure provides amethod of enhancing an immune response (e.g., enhanced T cell function,such as rescue from T cell functional exhaustion; enhanced Tcell-mediated response; increased inflammtary cytokine secretion and/orproduction, e.g., IFNγ secretion and/or production from T cells;enhanced NK cell function; enhanced macrophage function) in a subject inneed thereof, comprising administering to the subject a therapeuticallyeffective amount of a multispecific antigen-binding construct of thepresent disclosure. As exemplified herein, the enhancement of the immuneresponse is greater upon administration of the multispecificantigen-binding construct disclosed herein as compared to an agent(e.g., antibody) that binds either PD-1 or its ligand (e.g., PD-L1 orPD-L2), or a cocktail comprising an agent (e.g., antibody) that bindsPD-1 and an agent (e.g., antibody) that binds its ligand. In someembodiments, the enhancement of the immune response (e.g., enhanced Tcell function, such as rescue from T cell functional exhaustion;enhanced T cell-mediated response; increased inflammtary cytokine,IFNγsecretion and/or production from T cells; enhanced NK cell function;enhanced macrophage function) is greater by at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, or more, as compared to an agent(e.g., antibody) that binds either PD-1 or its ligand, or a cocktailcomprising an agent (e.g., antibody) that binds PD-1 and an agent (e.g.,antibody) that binds its ligand. Also provided herein are methods fortreating or delaying progression of a cancer or reducing or inhibitingtumor growth in a subject by administering to the subject an effectiveamount of a multispecific antigen-binding construct, an antibody orantigen-binding fragment thereof, a pharmaceutical composition, or aprotein conjugate as described herein.

The compositions described herein are useful in, inter alia, methods fortreating or preventing a variety of cancers in a subject.

The compositions can be administered to a subject, e.g., a humansubject, using a variety of methods that depend, in part, on the routeof administration. The route can be, e.g., intravenous injection orinfusion (IV), subcutaneous injection (SC), intraperitoneal (IP)injection, intramuscular injection (IM), or intrathecal injection (IT).The injection can be in a bolus or a continuous infusion.

As used herein, the term “subject” means a mammalian subject. Exemplarysubjects include, but are not limited to humans, monkeys, dogs, cats,mice, rats, cows, horses, camels, goats and sheep. In some embodiments,the subject is a human. In some embodiments, the subject has or issuspected to have a disease or condition that can be treated with amultispecific antigen-binding construct provided herein. In someembodiments, the disease or condition is a cancer. In some embodiments,the subject is a human with a cancer that can be treated with amultispecific antigen-binding construct provided herein. In someembodiments, the subject is a human that is suspected to have cancerthat can be treated with a multispecific antigen-binding constructprovided herein.

“Treating” or “treatment” of any disease or disorder refers, in someembodiments, to ameliorating a disease or disorder that exists in asubject. In another embodiment, “treating” or “treatment” includesameliorating at least one physical parameter, which can be indiscernibleby the subject. In yet another embodiment, “treating” or “treatment”includes modulating the disease or disorder, either physically (e.g.,stabilization of a discernible symptom) or physiologically (e.g.,stabilization of a physical parameter) or both. In yet anotherembodiment, “treating” or “treatment” includes delaying or preventingthe onset of the disease or disorder.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of a multispecificantigen-binding construct that, when administered to a subject, iseffective to treat a disease or disorder.

As used herein, “administer” or “administration” refers to the act ofinjecting or otherwise physically delivering a substance as it existsoutside the body (e.g., a multispecific antigen-binding constructprovided herein) into a patient, such as by mucosal, intradermal,intravenous, intramuscular delivery and/or any other method of physicaldelivery described herein or known in the art. When a disease, or asymptom thereof, is being treated, administration of the substancetypically occurs after the onset of the disease or symptoms thereof.When a disease, or symptoms thereof, are being prevented, administrationof the substance typically occurs before the onset of the disease orsymptoms thereof.

Administration can be achieved by, e.g., local infusion, injection, orby means of an implant. The implant can be of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. The implant can be configured for sustained or periodicrelease of the composition to the subject. See, e.g., U.S. PatentApplication Publication No. 20080241223; U.S. Pat. Nos. 5,501,856;4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures ofeach of which are incorporated herein by reference in their entirety.The composition can be delivered to the subject by way of an implantabledevice based on, e.g., diffusive, erodible, or convective systems, e.g.,osmotic pumps, biodegradable implants, electrodiffusion systems,electroosmosis systems, vapor pressure pumps, electrolytic pumps,effervescent pumps, piezoelectric pumps, erosion-based systems, orelectromechanical systems.

In some embodiments, a multispecific antigen-binding construct of thepresent disclosure is therapeutically delivered to a subject by way oflocal administration.

As used herein, the term “enhanced T cell function” or “activation of Tcells” refers to a cellular process in which mature T cells, whichexpress antigen-specific T cell receptors on their surfaces, recognizetheir cognate antigens and respond by entering the cell cycle, secretingcytokines or lytic enzymes, and initiating or becoming competent toperform cell-based effector functions. T cell activation requires atleast two signals to become fully activated. The first occurs afterengagement of the T cell antigen-specific receptor (TCR) by theantigen-major histocompatibility complex (MEW), and the second bysubsequent engagement of co-stimulatory molecules (e.g., CD28). Thesesignals are transmitted to the nucleus and result in clonal expansion ofT cells, upregulation of activation markers on the cell surface,differentiation into effector cells, induction of cytotoxicity orcytokine secretion, induction of apoptosis, or a combination thereof. Insome embodiments, “enhanced T cell function” also encompasses enhancedsurvival and/or enhanced proliferation of the T cell. Methods formeasuring such activities are routine and known in the art. In someembodiments, “enhanced T cell function” also encompasses rescue of a Tcell from an exhausted phenotype, so that restoration of or an increasein one or more T cell functions is achieved. As known in the art, thestate of T cell exhaustion is characterized by sequential loss of T celleffector functions, such as inflammtory cytokine production,proliferative abilities, metabolic fitness, in addition to sustainedupregulation of a wide array of co-inhibitory receptors, and uniquetranscriptional and epigenetic signatures. T cell exhaustion andalterations thereof can be measured using techniques known in the art,and described herein, for example, the in vitro nonspecific Tcell+K562-PD-L1 tumor target cell assay.

As used herein, the term T cell-mediated response refers to any responsemediated by T cells, including, but not limited to, effector T cells(e.g., CD8⁺ cells, effector γδ T cells) and helper T cells (e.g., CD4⁺cells, including subbsets thereof, such as T_(H)1, T_(H)2, T_(H)3,T_(H)17, T_(H)9, and T_(FH) cells). T cell-mediated responses include,for example, T cell cytotoxicity, T cell cytokine secretion, andproliferation. A suitable dose of an antibody or fragment thereofdescribed herein, which dose is capable of treating or preventing cancerin a subject, can depend on a variety of factors including, e.g., theage, sex, and weight of a subject to be treated and the particularinhibitor compound used. For example, a different dose of a wholemultispecific antigen-binding construct may be required to treat asubject with cancer as compared to the dose of a fragment of themultispecific antigen-binding construct (e.g., Fab′ antibody fragment)required to treat the same subject. Other factors affecting the doseadministered to the subject include, e.g., the type or severity of thecancer. For example, a subject having metastatic melanoma may requireadministration of a different dosage of multispecific antigen-bindingconstruct than a subject with glioblastoma. Other factors can include,e.g., other medical disorders concurrently or previously affecting thesubject, the general health of the subject, the genetic disposition ofthe subject, diet, time of administration, rate of excretion, drugcombination, and any other additional therapeutics that are administeredto the subject. It should also be understood that a specific dosage andtreatment regimen for any particular subject will also depend upon thejudgment of the treating medical practitioner (e.g., doctor or nurse).Suitable dosages are described herein. In some embodiments, themultispecific antigen-binding construct described herein are effectiveat both high and low doses.

A pharmaceutical composition can include a therapeutically effectiveamount multispecific antigen-binding construct described herein. Sucheffective amounts can be readily determined by one of ordinary skill inthe art based, in part, on the effect of the administered antibody, orthe combinatorial effect of the antibody and one or more additionalactive agents, if more than one agent is used. A therapeuticallyeffective amount of an antibody or fragment thereof described herein canalso vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody (and one ormore additional active agents) to elicit a desired response in theindividual, e.g., reduction in tumor growth. For example, atherapeutically effective amount of multispecific antigen-bindingconstruct can inhibit (lessen the severity of or eliminate theoccurrence of) and/or prevent a particular disorder, and/or any one ofthe symptoms of the particular disorder known in the art or describedherein. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

Suitable human doses of any of the multispecific antigen-bindingconstruct described herein can further be evaluated in, e.g., Phase Idose escalation studies. See, e.g., van Gurp et al. (2008) Am JTransplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res13(2, part 1):523-531; and Hetherington et al. (2006) AntimicrobialAgents and Chemotherapy 50(10): 3499-3500.

Toxicity and therapeutic efficacy of such compositions can be determinedby known pharmaceutical procedures in cell cultures or experimentalanimals (e.g., animal models of any of the cancers described herein).These procedures can be used, e.g., for determining the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. An antibody or antigen-binding fragment thereofthat exhibits a high therapeutic index is preferred. While compositionsthat exhibit toxic side effects may be used, care should be taken todesign a delivery system that targets such compounds to the site ofaffected tissue and to minimize potential damage to normal cells and,thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch antibodies or antigen-binding fragments thereof lies generallywithin a range of circulating concentrations of the antibodies orfragments that include the ED₅₀ with little or no toxicity. The dosagemay vary within this range depending upon the dosage form employed andthe route of administration utilized. For multispecific antigen-bindingconstruct described herein, the therapeutically effective dose can beestimated initially from cell culture assays. A dose can be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the EC₅₀ (i.e., the concentration of the construct—e.g.,antibody—which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography. Insome embodiments, e.g., where local administration (e.g., to the eye ora joint) is desired, cell culture or animal modeling can be used todetermine a dose required to achieve a therapeutically effectiveconcentration within the local site.

In some embodiments, the methods can be performed in conjunction withother therapies for cancer. For example, the composition can beadministered to a subject at the same time, prior to, or after,radiation, surgery, targeted or cytotoxic chemotherapy,chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, celltransplant therapy, precision medicine, genome editing therapy, or otherpharmacotherapy.

As described above, the multispecific antigen-binding constructdescribed herein be used to treat a variety of cancers selected from thegroup consisting of a hematological cancer, a lymphoma, a myeloma, aleukemia, a neurological cancer, skin cancer, breast cancer, a prostatecancer, a colorectal cancer, lung cancer, head and neck cancer, agastrointestinal cancer, liver cancer, pancreatic cancer, agenitourinary cancer, a bone cancer, renal cancer, and a vascularcancer. Optionally, the cancer is selected from the group consisting ofKaposi's sarcoma, leukemia, acute lymphocytic leukemia (etv6, aml1,cyclophilin b), acute myelocytic leukemia, myeloblasts promyelocytemyelomonocytic monocytic erythroleukemia, chronic leukemia, chronicmyelocytic (granulocytic) leukemia, chronic lymphocytic leukemia(cyclophilin b), mantle cell lymphoma, primary central nervous systemlymphoma, Burkitt's lymphoma, marginal zone B cell lymphoma(Ig-idiotype), Polycythemia vera Lymphoma, Hodgkin's disease (Imp-1,EBNA-1), non-Hodgkin's disease, mycloma (MUC family, p21ras), multiplemyeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solidtumors, sarcoma, carcinoma, fibrosarcoma, myxosarcoma, liposarcoma,chrondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colon carcinoma(p21ras, HER2/neu, c-erbB-2, MUC family), pancreatic cancer, breastcancer (MUC family, HER2/neu, c-erbB-2), ovarian cancer, prostate cancer(Prostate Specific Antigen (PSA) and its antigenic epitopes PSA-1,PSA-2, and PSA-3, PSMA, HER2/neu, c-erbB-2, ga733 glycoprotein),squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma (HER2/neu, c-erbB-2),hepatoma, hepatocellular cancer (a-fetoprotein), bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, uterine cancer, testicular tumor (NY-ESO-1), lung carcinoma,small cell lung carcinoma, non-small cell lung carcinoma (HER2/neu,c-erbB-2), bladder carcinoma, epithelial carcinoma, glioma (E-cadherin,α-catenin, β-catenin, γ-catenin, p120ctn), astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma (p5 protein, gp75,oncofetal antigen, GM2 and GD2 gangliosides, Melan-A/MART-1, cdc27,MAGE-3, p21ras, gp100), neuroblastoma, retinoblastoma, nasopharyngealcarcinoma (Imp-1, EBNA-1), esophageal carcinoma, basal cell carcinoma,biliary tract cancer (p21ras), bladder cancer (p21ras), bone cancer,brain and central nervous system (CNS) cancer, cervical carcinoma (p53,p21ras), choriocarcinoma (CEA), colorectal cancers (colorectalassociated antigen (CRC)-0017-1A/GA733, APC), connective tissue cancer,cancer of the digestive system, endometrial cancer, esophageal cancer,eye cancer, head and neck cancer, gastric cancer (HER2/neu, c-erbB-2,ga733 glycoprotein), epithelial cell cancer (cyclophilin b),intraepithelial neoplasm, kidney cancer, larynx cancer, liver cancer,lung cancer (small cell, large cell) (CEA, MAGE-3, NY-ESO-1), oralcavity cancer (for example lip, tongue, mouth, and pharynx cancers),ovarian cancer (MUC family, HER2/neu, c-erbB-2), pancreatic cancer,rectal cancer, cancer of the respiratory system, skin cancer, thyroidcancer, and cancer of the urinary system.

In some embodiments, a multispecific antigen-binding construct describedherein can be administered to a subject as a monotherapy. Alternatively,as described above, the antibody or fragment thereof can be administeredto a subject as a combination therapy with another treatment, e.g.,another treatment for a cancer. For example, the combination therapy caninclude administering to the subject (e.g., a human patient) one or moreadditional agents that provide a therapeutic benefit to a subject whohas, or is at risk of developing, cancer. Chemotherapeutic agentssuitable for co-administration with compositions of the presentinvention include, for example: taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxyanthrancindione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Furtheragents include, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g. mechlorethamine, thioTEPA,chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, cis-dichlordiamine platinum (II)(DDP), procarbazine, altretamine,cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, ortriplatin tetranitrate), anthracycline (e.g. daunorubicin (formerlydaunomycin) and doxorubicin), antibiotics (e.g. dactinomcin (formerlyactinomycin), bleomycin, mithramycin, and anthramycin (AMC)), andanti-mitotic agents (e.g. vincristine and vinblastine) and temozolomide.In some embodiments, the multispecific antigen-binding construct and theone or more additional active agents are administered at the same time.In other embodiments, the multispecific antigen-binding construct isadministered first in time and the one or more additional active agentsare administered second in time. In some embodiments, the one or moreadditional active agents are administered first in time and themultispecific antigen-binding construct is administered second in time.

A multispecific antigen-binding construct described herein can replaceor augment a previously or currently administered therapy. For example,upon treating with a multispecific antigen-binding construct,administration of the one or more additional active agents can cease ordiminish, e.g., be administered at lower levels or dosages. In someembodiments, administration of the previous therapy can be maintained.In some embodiments, a previous therapy will be maintained until thelevel of the multispecific antigen-binding construct reaches a levelsufficient to provide a therapeutic effect. The two therapies can beadministered in combination.

Monitoring a subject (e.g., a human patient) for an improvement in acancer, as defined herein, means evaluating the subject for a change ina disease parameter, e.g., a reduction in tumor growth. In someembodiments, the evaluation is performed at least one (1) hour, e.g., atleast 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration.The subject can be evaluated in one or more of the following periods:prior to beginning of treatment; during the treatment; or after one ormore elements of the treatment have been administered. Evaluation caninclude evaluating the need for further treatment, e.g., evaluatingwhether a dosage, frequency of administration, or duration of treatmentshould be altered. It can also include evaluating the need to add ordrop a selected therapeutic modality, e.g., adding or dropping any ofthe treatments for a cancer described herein.

In some embodiments, a multispecific antigen-binding construct describedherein is administered to modulate a T-cell response in a patient, forexample, by increasing T-cell activation and/or proliferation. Blockingthe interaction between PD-1 expressed by an immune cell and its ligandstrongly enhances T cell proliferation, IFNγ production and secretion,and the cytolytic activity of T cells. Bridging an immune cell thatexpresses PD-1 with a second cell (e.g., another immune cells, or atumor cell) that expresses a PD-1 ligand (e.g., PD-L1 or PD-L2) canstrongly enhance T cell proliferation, IFNγ production and secretion,and the cytolytic activity of T cells. Accordingly, in some embodiments,the multispecific antigen-binding construct of the present disclosure isadministered to a patient in need thereof to induce or increase T-cellactivation, enhance T cell proliferation, induce the production and/orsecretion of IFNγ, and/or induce a cytolytic T cell response.

EXAMPLES

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

Example 1: Induction of Interferon-Gamma (IFNγ) in T Cells

To assess the effect of PD1/PDL1 bispecific antibody on T-cellactivation, IFNγ production was analyzed in a mixed lymphocyte reaction(MLR). Aglycosylated bispecific antibodies combining binding domains ofNivolumabxAtezolizumab, 949×Atezolizumab, J43×Atezolizumab,PidilizumabxAtezolizumab, AtezolizumabxNivolumab, orDurvalumabxNivolumab were tested. The antibody 949 refers to a PD-1antibody as disclosed in U.S. Pat. No. 9,102,728. Antibody J43 refers toan anti-murine PD-1 antibody. KEYTRUDA, a humanized antibody that blocksPD-1 (Merck) and is known to induce IFNγ production, was used as acomparator. A schematic and amino acid sequence forNivolumabxAtezolizumab, 949 xAtezolizumab, and AtezolizumabxNivolumabare shown in FIGS. 4-6, respectively. Each of the bispecific formatsexemplified and tested herein were generated using known methods in theart. For example, as shown, in FIG. 4, the heavy chain portion of theAtezolizumab Fab was crosslinked to the heavy chain Fc portion ofNivolumab using methods known in the art. Suitable methods forcrosslinking two proteins, such as the heavy chain of a Fab and theheavy chain of IgG molecule, with or without linker sequences, aredescribed herein. This study demonstrated that various concentrations ofbispecific antibodies described above can induce an IFNγ response in Tcells.

Peripheral blood mononuclear cells (PBMCs) were isolated from leukopaks(HemaCare, Van Nuys, Calif.) derived from three independent human donors(D985, D7603, and D5004). Total T cells were enriched from PBMC bynegative selection using immunomagnetic cell separation (EASYSEP™;Stemcell Technologies, Vancouver BC). Monocytes were isolated from PBMCsusing immunomagnetic cell separation (EASYSEP™; Stemcell Technologies,Vancouver BC). T cells were resuspended in complete RPMI at 1×10⁶cells/ml concentration and monocytes were adjusted to 5×10⁵ cells/mlrespectively. In a 96-well plate, 100 μl of media containing T cellswere plated at 1×10⁵ cells/well density followed by adding 100 μl ofmonocyte cell suspension (E:T ratio 2:1). Next, 50 μl of mediacontaining various dilutions of antibodies was added to reach a finalconcentration of 0 nM, 0.5 nM, 5 nM, or 50 nM. Plates were incubated at37° C. in a CO₂ incubator for five days. At the end of incubationperiod, culture supernatants were collected and IFNγ levels wereanalyzed by MSD assay (Mesoscale Diagnostics, Rockville, Md.).

Example 2: Induction of Interferon-Gamma (IFNγ) in T Cells Treated witha Pembrolizumab/Atezolizumab Bispecific Antibody

To assess the effect of PD1/PDL1 bispecific antibody on T-cellactivation, IFNγ production was analyzed in a mixed lymphocyte reaction(MLR). A PembrolizumabxAtezolizumab bispecific, Pembrolizumab mAb,Atezolizumab Fab, a mix of KEYTRUDA and Atezolizumab Fab, and KEYTRUDA,a humanized antibody that blocks PD-1 (Merck) and is known to induceIFN-γ production, were used as comparators. A schematic and amino acidsequence for the PembrolizumabxAtezolizumab bispecific antibody is shownin FIG. 3. As similarly described for the other bispecific formatsexemplified and tested herein, the bispecific format shown in FIG. 3 wasgenerated using known methods in the art. As shown in FIG. 3, the heavychain portion of the Atezolizumab Fab was crosslinked to the c-terminusof the heavy chain Fc portion of Pembrolizumab using methods known inthe art. Suitable methods for crosslinking two proteins, such as theheavy chain of a Fab and the heavy chain of IgG molecule, with orwithout linker sequences, are described herein. These results indicatethat PembrolizumabxAtezolizumab, a bispecific antibody targeting PD-1and PD-L1, induces an IFNγ response in T cells comparable toPembrolizumab, Atezolizumab Fab, or KEYTRUDA treatments alone.

T cells were prepared as described above. A volume of 50 μl of mediacontaining various dilutions of antibodies was added to reach a finalconcentration of 0 nM, 0.5 nM, 5 nM, or 50 nM. Plates were incubated at37° C. in a CO₂ incubator for five days. At the end of incubationperiod, culture supernatants were collected and IFN-γ levels wereanalyzed by MSD assay (Mesoscale Diagnostics, Rockville, Md.).

Example 3: Comparison of Interferon-Gamma (IFNγ) Induction in T CellsTreated with a Bispecific Antibody Targeting PD-1 and PD-L1 or Cocktailof Monoclonal Antibodies Targeting PD-1/PD-L1

To assess the effect of PD-1×PD-L1 or PD-L1×PD-L1 bispecific antibodieson T-cell activation, IFN-γ production was analyzed in a mixedlymphocyte reaction (MLR). A PembrolizumabxAtezolizumab bispecificantibody, NivolumabxAtezolizumab bispecific antibody, a cocktail ofKEYTRUDA and Atezolizumab, a cocktail of Nivolumab and Atezolizumab weretested and KEYTRUDA alone, a humanized antibody that blocks PD-1 (Merck)and is known to induce IFN-γ production, was used as a comparator.

T cells were prepared as described above. A volume of 50 μl of mediacontaining various dilutions of antibodies was added to reach a finalconcentration of 0 nM, 0.01 nM, 0.001 nM, or 0.0001 nM. Plates wereincubated at 37° C. in a CO₂ incubator for five days. At the end ofincubation period, culture supernatants were collected and IFNγ levelswere analyzed by MSD assay (Mesoscale Diagnostics, Rockville, Md.).

FIG. 1 shows the concentration of IFN-γ as pg/mL at the finalconcentrations of antibodies tested, as indicated. These resultsindicate that bispecific PD-1×PD-L1 antibodies, such asPembrolizumabxAtezolizumab or the PDL1/PDL1 bispecific antibodyNivolumab/Atezolizumab, induce a higher IFNγ response in T cells than acocktail of PD1 and PDL1 specific antibodies, or a PD-1 antibody(KEYTRUDA) alone.

Example 4: Comparison of Interferon-Gamma (IFNγ) Induction in T CellsTreated with a Bispecific Antibody Targeting PD-1/PDL-1 or MonoclonalAntibodies Targeting PD-1 or PD-L1

To assess the effect of a PD-1×PD-L1 or PD-L1×PD-L1 bispecific antibodyon T-cell activation, IFNγ production was analyzed in a mixed lymphocytereaction (MLR). PembrolizumabxNivolumab bispecific antibody,AtezolizumabxAtezolizumab tetravalent, monospecific antibody were testedand Nivolumab alone, Atezolizumab alone, and KEYTRUDA (Pembrolizumab), ahumanized antibody that blocks PD-1 (Merck) and is known to induce IFN-yproduction, were used as comparators.

T cells were prepared as described above. A volume of 50 μl of mediacontaining various dilutions of antibodies was added to reach a finalconcentration of 0 nM, 0.01 nM, 0.001 nM, or 0.0001 nM. Plates wereincubated at 37° C. in a CO₂ incubator for five days. At the end ofincubation period, culture supernatants were collected and IFN-γ levelswere analyzed by MSD assay (Mesoscale Diagnostics, Rockville, Md.).

FIG. 2 shows the concentration of IFN-γ as pg/mL at the finalconcentrations of antibodies tested, in PBS, as indicated. These resultsindicate that bispecific PD1×PDL1 (PembrolizumabxNivolumab) orPD-L1×PD-L1 antibodies (AtezolizumabxAtezolizumab) induce a similarIFN-γ response in T cells as PD-1 (Atezolizumab or KEYTRUDA) or PD-L1(Nivolumab) specific antibodies alone.

FIG. 7 shows the concentration of IFN-γ as pg/mL at the finalconcentrations of antibodies tested, in PBS, as indicated. These resultsindicate that bispecific antibodies PD-1×PD-L1(PembrolizumabxAtezolizumab) or (NivolumabxAtezolizumab) in amultispecific format induce a greater IFN-γ response at femtomolarconcentrations in a mixed lymphocyte reaction (MLR) as compared to acocktail of Pembrolizumab and Atezolizumab or Nivolumab andAtezolizumab. These results suggest a synergistic effect that resultsfrom a multispecific format.

FIG. 8 shows an exemplary workflow for identifying multispecific (e.g.,bispecific) antibodies that demonstrate synergy. The process includes anunbiased screen of checkpoint blocker combinations in mixed lymphocytereaction (MLR), which measures IFNγ release, in pg/mL, at variousconcentrations, as described elsewhere herein (see, for example, thesection below entitled Nonspecific T cell+K562-PD-L1 tumor target cellassay). In the second step of the illustrated workflow, common lightchain bispecifics were generated to further test their efficacy andvarious exemplary common light chain bispecific formats are depicted.The identified common light chain bispecific formats outperform knownPD-1 blockers in a T-cell activation assay.

Example 5: Generation and Characterization of Anti-PD-1 and Anti-PD-L1Antibody Constructs and Multispecific Antigen-Binding Molecules AffinityMaturation of Anti-PD-L1 Antibodies

Affinity matured anti-PD-L1 antibodies derived from PD-L1 antibody mAb24were generated via construction of a mutant library, mammalian displaysorting, and screening of monoclonal IgG. The library containedmutations in the heavy chain, where synthetic diversity in CDRH1, CDRH2,and CDRH3 was introduced, while the light chain sequence was heldconstant, except in some cases, to maintain compatibility with singlelight chain constructs. The library went through 3 rounds of mammaliandisplay sorting aimed at increasing affinity for human PD-L1 andmaintaining mouse cross-reactivity. In each round, an off-ratecompetition step was employed after initial binding to biotinylatedantigens (i.e., 1 hour incubation with excess unlabeled antigen orparental IgG). After the final round of sorting, clones were picked,their sequences analyzed, and unique clones were assayed via Wasatch SPRbinding kinetics and cell-binding equilibrium assays to identify leadcandidates.

The resulting anti-PD-L1 antibodies from different selection rounds wereplotted on k_(d)/k_(a) double log plots. Apparent association anddissociation kinetic rate constants (k_(a) and k_(d) values) weredetermined on an SPRi reader (MX96, Carterra, Salt Lake City, Utah)) ina running buffer of PBS-Tween 0.01%. Anti-human PD-L1 antibodies werecovalently conjugated on a Carboxymethyldextran hydrogel 50 L chip(XanTec bioanalytics GmbH, Dusseldorf, Germany) on a CFM (Carterra).Freshly mixed activating reagents (150 ul of 0.4 M EDC and 150 ul of 0.1M sulfo-NHS in 5 ml of H2O) were used to activate the surface of the SPRsubstrate for 7 minutes. Antibodies at 10 mg/ml, in acetic acid bufferpH 4.5, were used for printing for 15 minutes. The printed chip was thenquenched on an SPRi reader (MX96, Carterra) with 1 M ethanolamine for 15minutes. For kinetics analysis, purified recombinant His tagged humanPD-L1 (0, 2.05, 5.12, 12.8, 32, 80, 200, 500 nM) was injectedsequentially. For each concentration, there was 5 minutes of associationfollowed by 10 minutes of dissociation. Data were processed and analyzedin SPR Inspection Tool and Scrubber softwares (Biosensor Tools LLC, SaltLake City, Utah). The kinetic data were referenced with the interstitialreference spots and double-referenced to a buffer cycle, and then fitglobally to a 1:1 binding model to determine their parent associationand dissociation kinetic rate constants (k_(a) and k_(d) values). Theratio k_(a)/k_(a) was used to derive the K_(D) value of each antigen/mAbinteraction, i.e. K_(D)=k_(d)/k_(a).

Affinity Maturation of Anti-PD-1 Antibodies

Affinity matured anti-PD-1 antibodies derived from PD-1 antibody mAb25were generated via construction of a mutant library, phage displaypanning, and screening of monoclonal antibodies. The library containedmutations in the heavy chain, where synthetic diversity in CDRH1, CDRH2,and CDRH3 was introduced, while the light chain sequence was heldconstant to maintain compatibility with single light chain constructs.The library went through 4 rounds of phage display panning rounds aimedat increasing affinity for human PD-1 and gaining mousecross-reactivity. In each round, an off-rate competition step wasemployed after initial binding to biotinylated antigens (i.e., 1 hourincubation with excess unlabeled antigen or parental IgG). After thefinal round of panning, clones were picked, their sequences analyzed,and unique Fab clones were assayed via Octet SPR binding kinetics andcell-binding equilibrium assays to identify lead candidates. Leadcandidates were converted from Fab to human IgG and furthercharacterized.

The resulting anti-PD-1 antibodies from different selection rounds wereplotted on k_(d)/k_(a) double log plots. Apparent association anddissociation kinetic rate constants (k_(a) and k_(d) values) weredetermined on an SPRi reader (MX96, Carterra, Salt Lake City, Utah)) ina running buffer of PBS-Tween 0.01%. Anti-human PD-1 antibodies werecovalently conjugated on a Carboxymethyldextran hydrogel 50 L chip(XanTec bioanalytics GmbH, Dusseldorf, Germany) on a CFM (Carterra).Freshly mixed activating reagents (150 ul of 0.4 M EDC and 150 ul of 0.1M sulfo-NHS in 5 ml of H2O) were used to activate the surface of the SPRsubstrate for 7 minutes. Antibodies at 10 mg/ml, in acetic acid bufferpH 4.5, were used for printing for 15 minutes. The printed chip was thenquenched on an SPRi reader (MX96, Carterra) with 1 M ethanolamine for 15minutes. For kinetics analysis, purified recombinant His tagged humanPD-1 (0, 2.05, 5.12, 12.8, 32, 80, 200, 500 nM) was injectedsequentially. For each concentration, there was 5 minutes of associationfollowed by 10 minutes of dissociation. Data were processed and analyzedin SPR Inspection Tool and Scrubber softwares (Biosensor Tools LLC, SaltLake City, Utah). The kinetic data were referenced with the interstitialreference spots and double-referenced to a buffer cycle, and then fitglobally to a 1:1 binding model to determine their apparent associationand dissociation kinetic rate constants (k_(a) and k_(d) values). Theratio k_(d)/k_(a) was used to derive the K_(D) value of each antigen/mAbinteraction, i.e. K_(D)=k_(d)/k_(a).

Nonspecific T Cell+K562-PD-L1 Tumor Target Cell Assay

T cells were isolated from previously frozen PBMCs (peripheral bloodmononuclear cells) using a negative selection kit and activated usingIMMUNOCULT™ anti-CD3/CD28 T cell activator in X-VIVO 15 mediasupplemented with 10% FBS (“X-10”). After 3 days, the cells wereswitched to X-10 media containing 5 ng/ml IL-2 and 2.5 ng/ml IL-7(“hX-10”). Every 2-3 days the cells were fed fresh hX-10. After 10 daysof activation, the IMMUNOCULT™-expanded T cells were CELLTRACE™Violet-labeled and co-cultured with K562 cells that were stablytransduced to express human CD32B and PD-L1 (“K32P”) in 96 wellround-bottom plates with 50,000 T cells and 25,000 K32P cells per well.Antibodies (Bispecific 3, KEYTRUDA, and an isotype control) were addedat final concentrations between 10 and 0.001 nM done in 10-folddilutions along with 0.25 μg/ml anti-CD3 (clone OKT3). After 3 days,supernatants were collected for measuring IFNγ cytokine production viaMSD plates, and cells were stained for flow cytometry, and then run on aBD LSRFORTESSA™ cytometer to look at T cell activation and target cellkilling. Target cell killing is measured by counting the number of liveK32P targets in the experimental wells compared to the number of K32Ptargets in a set of no CD3 control wells. All data was then analyzed inGraphPad Prism. As shown in FIGS. 9A-9B, Bispecific 3 induced higheramounts of IFNγ and killing of K32P target cells as compared to both theisotype control antibody and KEYTRUDA.

CMV Antigen Recall Assay Day 12 expanded CMV antigen specific CD8+ Tcells, from an HLA A02:01 donor, were thawed and rested overnight inhX-10 media containing 2 ug/ml DNase I. The following day, the cellswere collected, and dead cells were removed using Ficoll-separation. Theremaining cells were then co-cultured with K562 cells expressingHLA-A02:01, CMV protein pp65-IRES-GFP, and PD-L1 (“KACP”, GFP+) and K562cells expressing HLA-A02:01 (“KA”, GFP-) in 96 well round-bottom plateswith 25,000 CMV T cells, 50,000 KACP cells, and 50,000 KA cells perwell. Antibodies (Bispecific 3, KEYTRUDA, a combination of mAb1 andmAb28, and an isotype control) were added at final concentrationsbetween 10 and 0.0001 nM. This dosing included 10-fold dilutions between10 and 0.1 nM and then 2-fold dilutions until 0.0001 nM. After 2 days ofco-culture, specific killing of KACP tumor targets via flow cytometrywere analyzed. Specific killing is defined ratiometrically as the ratioof live GFP+ KACP cells to GFP-KA cells, normalized to the ratio ofthese cells when no T cells are present. As shown in FIG. 10A,Bispecific 3 increased the specific killing of tumor antigen targetcells. The increase mediated by Bispecific 3 was always higher than whatwas seen for KEYTRUDA, while at high doses the combination of mAb1 andmAb28 killed an equal number of KACP cells. Critically, at the low dosesof antibody (0.001 through 0.01 nM) Bispecific 3 showed an increase inkilling of KACP cells as compared to both KEYTRUDA and the combinationof mAb1 and mAb28, indicating that Bispecific 3 can be used to mediateantigen-specific killing of target cells at lower doses.

In a separate experiment, the effect of Bispecific 3 on Rajicell-specific killing was examined in a manner similar to that outlinedabove for K562 cell-specific killing assay. Briefly, CMV antigenspecific CD8+ T cells were co-cultured with Raji cells expressingHLA-A02:01, CMV protein pp65-IRES-GFP, and PD-L1 (“RACP”, GFP+) and Rajicells expressing HLA-A02:01 (“RA”, GFP-) in 96 well round-bottom plateswith 25,000 CMV T cells, 50,000 RACP cells, and 50,000 RA cells perwell. Antibodies (Bispecific 3, KEYTRUDA, a combination of mAb1 andmAb28, and an isotype control) were added at final concentrationsbetween 10 and 0.0001 nM. This dosing included 10-fold dilutions between10 and 0.1 nM and then 2-fold dilutions until 0.0001 nM. After 2 days ofco-culture, specific killing of RACP tumor targets via flow cytometrywere analyzed. Specific killing is defined ratiometrically as the ratioof live GFP+ RACP cells to GFP− RA cells, normalized to the ratio ofthese cells when no T cells are present. As shown in FIG. 10B,Bispecific 3 increased the specific killing of tumor antigen targetcells. The increase mediated by Bispecific 3 was noticeably higher at0.001 nM than what was seen for either KEYTRUDA or the combination ofmAb1 and mAb28, while at high doses Bispecific 3 and the combination ofmAb1 and mAb28 caused comparable killing of target cells. These dataindicate that Bispecific 3 can be used to mediate antigen-specifickilling of target cells at lower doses.

Staphylococcus aureus Enterotoxin A (“SEA”) Assay

Previously frozen PBMCs were thawed and incubated with 0.1 mg/ml DNase Iin PBS for 15 minutes, passed through a 40 μm nylon mesh filter, andthen plated in X-10 media at 100,000 cells per well of a 96 wellround-bottom plate. Antibodies (Bispecific 3, KEYTRUDA, a combination ofmAb1 and mAb28, and an isotype control) were added at finalconcentrations between 10 and 0.0001 nM, along with 10 ng/ml SEA. After3 days of co-culture, IL-2 cytokine production was analyzed. As shown inFIG. 11, Bispecific 3 induced more IL-2 than KEYTRUDA at all testeddoses. Importantly, Bispecific 3 induced increased IL-2 productionstarting at lower concentrations of antibody as compared to bothKEYTRUDA and the combination of mAb1 and mAb28.

PD-1 Expression Determination Assays

Previously frozen PBMCs were thawed and incubated with 0.1 mg/ml DNase Iin PBS for 15 minutes, passed through a filter, and then plated at 1×10⁶cells/ml in hX-10 with 0.25 μg/ml anti-CD3 (clone OKT3) and 0.25 μg/mlanti-CD28 (clone CD28.2) for 3 days. After 3 days, the cells wereadjusted to 2×10⁶ cells/ml in hX-10 by spinning down the cells andremoving excess media. The cells were then resuspended and plated at1×10⁶ cells/well (0.5 ml) in a 48 well plate. Antibodies (Bispecific 3,KEYTRUDA, a combination of mAb1 and mAb28, Atezolizumab, or isotypecontrol) were then added to each well to get a final volume of 1 ml/wellhX-10, with final concentrations of 0.01 nM or 1 nM antibody. After anovernight culture, the cells were collected into 1.5 ml Eppendorf tubesand lysed in 100 ul of Lysis buffer containing a protease inhibitorcocktail. After spinning to clear the supernatants of particulates,cleared supernatant was stored at −80° C. until use for western blots.For Western blots, samples were adjusted to include 1×LDS sample bufferand 1× reducing agent, heated at 70° C. for 10 minutes, then 20 ul perwell was loaded onto 4-12% Bis-Tris gels. After the gel ran, it wastransferred to nitrocellulose membrane, blocked with TBS-0.1% Tween-20(TBST) containing 5% dry milk for 1 hour at room temperature, washed inTBST, and then incubated overnight at 4° C. with anti-PD-1 (clone D4W2J,Cell Signaling Technologies) or anti-β-Actin (Clone 13E5, Cell SignalingTechnologies) antibodies in TBST containing 5% bovine serum albumin. Thefollowing day, the membranes were washed in TBST, incubated with aHRP-conjugated anti-Rabbit IgG antibody, washed in TBST, then developedwith SUPERSIGNAL™ Pico substrate. Chemiluminescent and white lightimages were collected on the Amersham Imager 600 and superimposed togenerate the images shown. As shown in FIG. 12A, Bispecific 3 is uniquein its ability to cause the loss of cellular PD-1 expression byinternalization and/or subsequent degradation of PD-1 and/or shedding.

In a subsequent assay, previously frozen PBMCs were thawed and incubatedwith 0.1 mg/ml DNase I in PBS for 15 minutes, passed through a filter,and then plated at 1×10⁶ cells/ml in hX-10 with 0.25 μg/ml anti-CD3(clone OKT3) and 0.25 μg/ml anti-CD28 (clone CD28.2) for 3 days. After 3days, the cells were adjusted to 2×10⁶ cells/ml in hX-10 by spinningdown the cells and removing excess media. The cells were thenresuspended and plated at 1×10⁶ cells/well (0.5 ml) in a 48 well plate.Antibodies (Bispecific 3, KEYTRUDA, a combination of mAb1 and mAb 28,Atezolizumab, Atezolizumab and KEYTRUDA, Bispecific 3 with 50 nM mAb 1,or isotype control) were then added to each well to get a final volumeof 1 ml/well hX-10 with final concentration of 0.1 nM, 1 nM, or 10 nMantibody. After an overnight culture, the cells were collected into 1.5ml Eppendorf tubes and lysed in 100 ul of Lysis buffer containing aprotease inhibitor cocktail. After spinning to clear the supernatants ofparticulates, cleared supernatant was stored at −80° C. until use forwestern blots. For western blots, samples were adjusted to include 1×LDSsample buffer and 1× reducing agent, heated at 70° C. for 10 minutes,then 10 ul per well was loaded onto 4-12% Bis-Tris gels. After the gelran, it was transferred to nitrocellulose membrane, blocked withTBS-0.1% Tween-20 (TBST) containing 5% dry milk for 1 hour at roomtemperature, washed in TBST, and then incubated overnight at 4 C withanti-PD-1 (clone D4W2J, Cell Signaling Technologies) or anti-β-Actin(Clone 13E5, Cell Signaling Technologies) antibodies TBST containing 5%bovine serum albumin. The following day, the membranes were washed inTBST, incubated with a HRP-conjugated anti-Rabbit IgG antibody, washedin TBST, then developed with SUPERSIGNAL™ Pico substrate.Chemiluminescent and white light images were collected on the AmershamImager 600 and superimposed to generate the images shown. As shown inFIGS. 12B-12C, Bispecific 3 is unique in its ability to cause the lossof cell-surface PD-1 expression through internalization and/orsubsequent degradation of PD-1 and/or shedding of PD-1. Additionally,when the anti-PD-L1 antibody, mAb1, was added at 50 nM to wellscontaining Bispecific 3, the ability of Bispecific 3 to drive loss ofcell-surface PD-1 expression by PD-1 internalization and/or subsequentdegradation of PD-1 and/or shedding was lost. This suggests that botharms of Bispecific 3 should be engaged to drive loss of cell-surfacePD-1 expression by internalization and/or degradation of PD-1 and/orshedding.

FIG. 12D shows that treatment with Bispecific 3 increases the amount ofPD-1 in the supernatant when both binding arms of the bispecific areengaged concurrently. This effect is lost when the PD-L1 targeting armis blocked by mAb1, the parent PD-L1 arm antibody. This suggests thatBispecific 3 increases shedding of PD-1 into the supernatant.

The effect of valency versus the sequence of the binding arms was nextinvestigated. A new bispecific was generated (Bispecific 5) having afirst N-terminal Fab that binds PD-L1 derived from the VH and VLsequences of mAb1, and a second N-terminal Fab that binds PD-1 derivedfrom the VH and VL sequences of mAb28. In other words, while they sharethe same VH and VL sequences for binding to PD-1 and PD-L1, Bispecific 5has one monovalent arm binding PD-L1 and one movalent arm binding PD-1,as compared to Bispecific 3, which has bivalent arms binding PD-L1 andbivalent arms binding PD-1.

40×10⁶ PBMCs were treated with 0.25 μg/ml anti-CD3 and 0.25 μg/mlanti-CD28 for 3 days in a T75 flask in hX-10 media containing IL-2 andIL-7. Cells were incubated for 3 days in the flask with no manipulation.On Day 3, the cells were collected without washing away any of the oldmedia. Cells were plated in wells of a 48-well plate, at a final volumeof 1 ml, with 1×10⁶ cells/well and antibodies at 0.01 nM, 0.1 nM, 1 nM,and 10 nM. After an overnight incubation, cells were collected into 1.5ml tubes, with a wash to collect all cells from the well, then lysed andused for western blotting. Western blots were run for PD-1 and Actin.FIG. 12E demonstrates that the valency of the binding arms influencesthe degree of the loss of PD-1 expression. As shown, loss of PD-1expression starts to occur at higher doses of Bispecific 5 (monovalentbinding arms, total valency=2 or bivalent) versus Bispecific 3 (bivalentbinding arms, total valency=4 or tetravalent), suggesting that theincreased valency of Bispecific 3 is responsible for this difference.

Next, the effect(s) of ADAM/MMP inhibition was examined usingBatimastat, a broad-spectrum inhibitor of multiple MMPs and ADAMs, whichare sheddases or proteases responsible for cleaving proteins off theplasma membrane of cells. Briefly, 40×10⁶ PBMCs were treated with 0.25μg/ml anti-CD3 and 0.25 μg/ml anti-CD28 for 3 days in a T75 flask inhX-10 media containing IL-2 and IL-7. Cells were incubated for 3 days inthe flask with no manipulation. On Day 3, the cells were collectedwithout washing away any of the old media. Cells were plated in wells ofa 48-well plate, at a final volume of 1 ml, with 1×10⁶ cells/well. Atleast ½ hour before antibody addition, Batimastat or DMSO vehicle wereadded at increasing concentrations of 0 μM, 1.25 μM, 2.5 μM, 5 μM, and10 μM and pre-incubated at 37 C to look at the consequence of ADAM/MMPinhibition. Isotype control and Bispecific 3 were then added at 1 nM.After an overnight incubation, cells were collected into 1.5 ml tubes,with a wash to collect all cells from the well, then lysed and used forwestern blotting. Western blots were run for PD-1 and Actin. FIG. 12Fdemonstrates that pretreatment with Batimastat, a broad-spectruminhibitor of multiple MMPs and ADAMs, greatly reduces the amount ofcell-associated PD-1 loss, suggesting that PD-1 loss or shedding is dueto cleavage by an MMP or ADAM protease. FIG. 12G suggests thatBispecific 3 drives loss of cell-surface PD-1 expression primarily whenit binds to PD-1 and PD-L1 that are in the trans configuration, i.e.,are being expressed by different cells. Given that PD-1 and PD-L1 can beexpressed on the same cell, it was investigated whether Bispecific 3binding to PD-1 and PD-L1 in cis results in loss of PD-1 expression orPD-1 shedding, or whether the binding by Bispecific 3 needs to be intrans, with Bispecific 3 bridging a first cell, such as a tumor cellexpressing PD-L1, and a second cell, such as aT effector cell expressingPD-1. Jurkat cells expressing only PD-1, only PD-L1, or both PD-1 andPD-L1 were used in experiments. Briefly, a total of 0.5×10⁶ Jurkat cellsexpressing PD-1, Jurkat cells expressing PD-1 and PD-L1, or a 1:1 mix ofPD-1-only or PD-L1 only-expressing Jurkat cells were treated withisotype control or Bispecific 3 at 0.01, 0.1 and 1 nM. After anovernight incubation, cells were collected into 1.5 ml tubes, with awash to collect all cells from the well, then lysed and used for westernblotting. Western blots were run to determine PD-1 and Actin levels.

In Vivo Function of Bispecific 3

Day 13 expanded CMV antigen specific CD8+ T cells from an HLA A02:01donor were collected and then mixed with K562 cells expressingHLA-A02:01, CMV protein pp65-IRES-GFP, and PD-L1 (“KACP”) at a ratio of2:1 KACP:CMV T cell and Matrigel such that 100 μl included 1× Matrigel,5×10⁶ KACP cells, and 2.5×10⁶ CMV T cells. NSG mice were implantedsubcutaneously on their flank with 100 ul of prepared Matrigel-KACP-CMVT cell mixture per mouse. Antibody dosing with was started on the day ofimplantation and given at equimolar amounts (200 μg for each monoclonal,333 μg for Bispecific 3) and mice were re-dosed with antibody every 3days. Treatment groups included a control group that was given KACPtumor cells in Matrigel alone (“No T cell transfer”), and groups givenisotype, KEYTRUDA, mAb1 and mAb28 (mAb1+mAb28), or Bispecific 3. Therewere 5 mice per group, and mice were monitored daily, with tumorsmeasured twice a week. The protocol is schematically depicted in FIG.13A. As shown in FIG. 13B, the No T cell transfer group had tumors thatgrew more aggressively than any group containing T cells. In this model,KEYTRUDA gave no benefit in delaying KACP tumor growth as compared tothe isotype control. Both Bispecific 3 and the combination mAb1 andmAb28 groups had significant delays in tumor growth as compared to boththe isotype and KEYTRUDA groups. At day 24, there was a significantdivergence between the Bispecific 3 group and the group treated with acombination of mAb1 and mAb28, with Bispecific 3 causing a delay intumor growth as compared to the combination.

Example 6: In Vivo Studies for Pharmacological Investigation ofBispecific 3

Co-Inoculation Model of K562-A2-CMV-PD-L1 Target Cells and CMV T-Cellsin NSG Mice

In two separate studies with the protocol schematically depicted in FIG.13A, NSG female mice were co-injected subcutaneously (s.c.) with 100 μLMatrigel containing 5×10⁶ K562-A2-CMV-PD-L1 (“KACP”) cells and either2.5×10⁶ (FIGS. 13B and 14A; effector: target ratio=0.5:1) or 5×10⁶ (FIG.14B; effector: target ratio=1:1) CMV-specific T cells expanded in vitrofrom the same donor. Mice were blind-grouped the day of inoculation in 5groups of 5 mice per group, or 10 mice per group respectively. Todetermine the anti-tumor activity of the human T cells, in both studiesthe first group of mice were injected only with tumor cells. The secondgroup received human IgG1 isotype control antibodies (0.2 mg Q3D×5). Thethird group received KEYTRUDA (0.2 mg Q3D×5), the fourth group wastreated with Bispecific 3 (0.333 mg Q3D×5), and the fifth group receiveda combination of the anti-PD-1 (mAb28) and anti-PD-L1 (mAb1) antibodies(0.2 mg each Q3D×5). All antibodies were injected i.p. and dosingstarted on implantation day. Anti-tumor activity was determined by tumorgrowth monitored by tumor volume measurements, body weight loss, andoverall survival. Tumor size and body weight were measured 2-3 times perweek, with mice euthanized when tumors were approaching 2000 mm³ or micelost 20% of body weight. Data was analyzed and graphed using Graph PadPrism software. As illustrated in FIGS. 14A and 14B, Bispecific 3displayed increased anti-tumor efficacy as compared to the differentmonoclonal antibodies tested.

Transplantable Syngeneic Mouse Models

Breast cancer EMT-6 cells (5×10⁴/mouse) were implanted in the mammaryfat pad of BALB/c female mice. When tumors were established (tumor volapproximately 50 mm³), mice were grouped (n=8) and treated with humanIgG1 isotype control (0.2 mg Q3D×3) or Bispecific 3 (0.333 mg Q3D×3).Antibodies were delivered by i.p. injections.

Using a similar study design, bladder cancer MB-49 cells (5×10⁵/mouse)were injected s.c. in female C57BL/6 mice. When tumors were established(tumor volume approximately 75 mm³) mice were grouped (n=8) and treatedwith human IgG1 isotype control (0.2 mg Q3D×3) or Bispecific 3 (0.333 mgQ3D×3). Antibodies were delivered by i.p. injections.

For both studies, tumor size and body weight were measured 2-3 times perweek, with mice euthanized when tumors were approaching 2000 mm³ or micelost 20% of body weight. Results were plotted, graphed, and analyzed byGraph Pad Prism software. Bispecific 3 showed greater anti-tumorefficacy in the EMT-6 breast cancer cell model (FIG. 15A) and in theMB-49 cell model (FIG. 15B) as compared to the isotypecontrol-treatment.

Engineered Transplantable Syngeneic Models in Transgenic Mice

Mouse colon cancer MC-38 cells engineered to express human PD-L1(MC-38-hPD-L1) were injected s.c. in C57BL/6 female mice in which theextracellular domains of PD-1 and PD-L1 were replaced with human PD-1and PD-L1, while the transmembrane and signaling domains of thereceptor-ligand pair were not modified. The genetic knock-in of humanPD-1 and PD-L1 allowed for testing our bispecific PD-1×PD-L1 antibodieshead to head with KEYTRUDA, which does not interact with mouse PD-1, andthus, cannot be evaluated in syngeneic mouse models. Mice withestablished MC-38-hPD-L1 tumors were grouped (n=8) and treated i.p. withisotype control (0.2 mg Q3D×3), KEYTRUDA (0.2 mg Q3D×3), or Bispecific 3(0.333 mg Q3D×3). Treatment with Bispecific 3 controlled MC-38-hPD-L1tumor growth significantly better than isotype control- or KEYTRUDAtreatments (FIG. 16A). In addition, Bispecific 3 treatment resulted inan increase in survival in the MC-38-hPD-L1 tumor mice as compared tothe isotype control- or KEYTRUDA-treated mice (FIG. 16B).

In a separate study, C57BL/6 human PD-1/PD-L1 transgenic female micewere injected s.c. with 1×10⁵ B16F10-hPD-L1 cells, an extremelyaggressive mouse melanoma cell line. Mice were grouped (n=8) as soon asthe melanoma was visible (day 4 after tumor cell inoculation) in 5groups which received the following treatment. Group 1 received isotypecontrol human IgG1 (0.2 mg Q3D×3), group 2 Avelumab (0.2 mg Q3D×3),group 3 KEYTRUDA (0.2 mg Q3D×3), group 4 Bispecific 3 (0.333 Q3D×3), andgroup 5 combination of KEYTRUDA and Avelumab (0.2 mg each Q3D×3). Forboth studies tumor size and body weight were measured 2-3 times perweek, and mice euthanized when tumors were approaching 2000 mm³ or micelost 20% of body weight. Survival was recorded and analyzed. Data wereplotted, graphed, and analyzed by Graph Pad Prism software. Asillustrated in FIGS. 17A and 17B, Bispecific 3 was significantly moreeffective in delaying tumor growth in the B16F10-hPD-L1 mice as comparedto the other treatment groups tested. In addition, B16F10-hPD-L1 micetreated with Bispecific 3 survived longer on average than mice receivingthe other tested treatments (FIG. 17C and FIG. 17E).

Example 7: Bispecific 3 has Monoclonal-Like DLP's and Parental-LikeBinding

FIGS. 18A-18D demonstrate that Bispecific 3 has drug-like properties(DLP's) similar to a well-behaved monoclonal antibody and maintainsparental PD-1 and PD-L1 binding. FIG. 18A shows that Bispecific 3 showssimilar binding to CHO cells expressing human PD-1 as parental clonemAb28 (top), and to CHO cells expressing human PD-L1 as parental clonemAb1 (bottom). FIG. 18B shows that Bispecific 3 shows similar binding toCHO cells expressing cynomolgus PD-1 as parental clone mAb28 (top), andto CHO cells expressing cynomolgus PD-L 1 as parental clone mAb1(bottom). FIG. 18C shows that Bispecific 3 shows similar binding to CHOcells expressing mouse PD-1 as parental clone mAb28 (top), and to CHOcells expressing mouse PD-L1 as parental clone mAb1 (bottom). FIG. 18Dshows a size-exclusion chromatography trace of Bispecific 3 afterProtein A chromatography (top) demonstrating a single peak with greaterthan 98% purity and a differential scanning fluorimetry (DSF) trace ofBispecific 3 (bottom) demonstrating that the molecule has high thermalstability.

SEQUENCES SEQ ID NO: 114 (GenBank accession numberNP_005009.2, UniProt Q15116-full-length human PD-1 precursor)mqipqapwpv vwavlqlgwr pgwfldspdr pwnpptfspallvvtegdna tftcsfsnts esfvlnwyrm spsnqtdklaafpedrsqpg qdcrfrvtql pngrdfhmsv vrarrndsgtylcgaislap kaqikeslra elrvterrae vptahpspsprpagqfqtlv vgvvggllgs lvllvwvlav icsraargtigarrtgqplk edpsavpvfs vdygeldfqw rektpeppvpcvpeqteyat ivfpsgmgts sparrgsadg prsaqplrpe dghcswplSEQ ID NO: 115 (GenBank accession numberNP_054862.1, UniProt Q9NZQ7-human PD-L1)mrifavfifm tywhllnaft vtvpkdlyvv eygsnmtieckfpvekqldl aalivyweme dkniiqfvhg eedlkvqhssyrqrarllkd qlslgnaalq itdvklqdag vyrcmisyggadykritvkv napynkinqr ilvvdpvtse heltcqaegypkaeviwtss dhqvlsgktt ttnskreekl fnvtstlrintttneifyct frrldpeenh taelvipelp lahppnerthlvilgaillc lgvaltfifr lrkgrmmdvk kcgiqdtnsk kqsdthleetSEQ ID NO: 116 (GenBank accession numberNP_079515.2, UniProt Q9BQ51-human PD-L2)miflllmlsl elqlhqiaal ftvtvpkely iiehgsnvtlecnfdtgshv nlgaitaslq kvendtsphr eratlleeqlplgkasfhip qvqvrdegqy qciiiygvaw dykyltlkvkasyrkinthi lkvpetdeve ltcqatgypl aevswpnvsvpantshsrtp eglyqvtsvl rlkpppgrnf scvfwnthvreltlasidlq sqmeprthpt wllhifipfc iiafifiatvialrkqlcqk lysskdttkr pvtttkrevn sai

1-274. (canceled)
 275. A multispecific antigen-binding constructcomprising at least two antigen-binding arms, wherein a first arm bindsPD-1 expressed by an immune cell, and a second arm binds a PD-1 ligandwhich is expressed by a second cell, wherein the multispecificantigen-binding construct blocks the interaction of PD-1 and PD-1ligand.
 276. The multispecific antigen-binding construct of claim 275,wherein the second cell is a tumor cell that expresses PD-L1.
 277. Themultispecific antigen-binding construct of claim 275, wherein the firstarm binds PD-1 and comprises: (a) a heavy chain variable regioncomprising (i) a CDRH1 comprising SEQ ID NO: 70 (FTFX₁X₂YAX₃X₄, whereinX₁=S, R, G, or N; X₂=D, S, N, A, R, or G; X₃=M or L; X₄=S, L, or N);(ii) a CDRH2 comprising SEQ ID NO: 71; and (iii) a CDRH3 comprising SEQID NO: 72 (ARGLDFIVGX₅TGNDY, wherein X₅=A, Y, or R); and (b) a lightchain variable region comprising: (i) a CDRL1 comprising SEQ ID NO: 9;(ii) a CDRL2 comprising SEQ ID NO: 5; and (iii) a CDRL3 comprising SEQID NO:
 10. 278. The multispecific antigen-binding construct of claim275, wherein the first arm binds PD-1 and: (a) CDRH1 of the first armcomprises SEQ ID NO: 73, CDRH2 of the first arm comprises SEQ ID NO: 71and CDRH3 of the first arm comprises SEQ ID NO: 74; (b) CDRH1 of thefirst arm comprises SEQ ID NO: 73, CDRH2 of the first arm comprises SEQID NO: 71 and CDRH3 of the first arm comprises SEQ ID NO: 75; (c) CDRH1of the first arm comprises SEQ ID NO: 76, CDRH2 of the first armcomprises SEQ ID NO: 71 and CDRH3 of the first arm comprises SEQ ID NO:75; (d) CDRH1 of the first arm comprises SEQ ID NO: 77, CDRH2 of thefirst arm comprises SEQ ID NO: 71 and CDRH3 of the first arm comprisesSEQ ID NO: 75; (e) CDRH1 of the first arm comprises SEQ ID NO: 78, CDRH2of the first arm comprises SEQ ID NO: 71 and CDRH3 of the first armcomprises SEQ ID NO: 75; (f) CDRH1 of the first arm comprises SEQ ID NO:79, CDRH2 of the first arm comprises SEQ ID NO: 71 and CDRH3 of thefirst arm comprises SEQ ID NO: 75; (g) CDRH1 of the first arm comprisesSEQ ID NO: 80, CDRH2 of the first arm comprises SEQ ID NO: 71 and CDRH3of the first arm comprises SEQ ID NO: 75; (h) CDRH1 of the first armcomprises SEQ ID NO: 81, CDRH2 of the first arm comprises SEQ ID NO: 71and CDRH3 of the first arm comprises SEQ ID NO: 75; (i) CDRH1 of thefirst arm comprises SEQ ID NO: 82, CDRH2 of the first arm comprises SEQID NO: 71 and CDRH3 of the first arm comprises SEQ ID NO: 75; (j) CDRH1of the first arm comprises SEQ ID NO: 83, CDRH2 of the first armcomprises SEQ ID NO: 71 and CDRH3 of the first arm comprises SEQ ID NO:74; (k) CDRH1 of the first arm comprises SEQ ID NO: 84, CDRH2 of thefirst arm comprises SEQ ID NO: 71 and CDRH3 of the first arm comprisesSEQ ID NO: 85; (l) CDRH1 of the first arm comprises SEQ ID NO: 86, CDRH2of the first arm comprises SEQ ID NO: 71 and CDRH3 of the first armcomprises SEQ ID NO: 85; or (m) CDRH1 of the first arm comprises SEQ IDNO: 80, CDRH2 of the first arm comprises SEQ ID NO: 71 and CDRH3 of thefirst arm comprises SEQ ID NO:
 85. 279. The multispecificantigen-binding construct of claim 275, wherein the first arm binds PD-1and the heavy chain variable region of the first arm comprises an aminoacid sequence that is at least 85% identical to any one of SEQ ID NOs:87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or
 99. 280. Themultispecific antigen-binding construct of claim 275, wherein the firstarm binds PD-1 and the heavy chain variable region of the first armcomprises an amino acid sequence that is at least 85% identical to SEQID NO:
 90. 281. The multispecific antigen-binding construct of claim279, wherein the first arm binds PD-1 and the light chain variableregion of the first arm comprises an amino acid sequence that is atleast 90% identical to SEQ ID NO:
 59. 282. The multispecificantigen-binding construct of claim 275, wherein the second arm bindsPD-L1 and comprises: a. a heavy chain variable region comprising (i) aCDRH1 comprising SEQ ID NO: 1; (ii) a CDRH2 comprising SEQ ID NO: 2(GGIIPX₁X₂GX₃ATYA, wherein X₁ is V or I; X₂ is F, L, or V; and X₃ is Tor A); and (iii) a CDRH3 comprising SEQ ID NO: 3 (ARLKX₁ELKDAFDI,wherein X₁ is G, F, or N); and b. a light chain variable regioncomprising: (i) a CDRL1 comprising SEQ ID NO: 4 (RASQX₁ISSYLN, whereinX₁ is S, W, or Q); (ii) a CDRL2 comprising SEQ ID NO: 5; and (iii) aCDRL3 comprising SEQ ID NO: 6 (X₁QSYSTPLT, wherein X₁ is Q or F). 283.The multispecific antigen-binding construct of claim 275, wherein thesecond arm binds PD-L1 and CDRH1 of the second arm comprises SEQ ID NO:1, CDRH2 of the second arm comprises SEQ ID NO: 7 and CDRH3 of thesecond arm comprises SEQ ID NO:
 8. 284. The multispecificantigen-binding construct of claim 283, wherein the second arm bindsPD-L1 and CDRH1 of the second arm comprises SEQ ID NO: 1, CDRH2 of thesecond arm comprises SEQ ID NO: 7, CDRH3 of the second arm comprises SEQID NO: 8; CDRL1 of the second arm comprises SEQ ID NO: 9; CDRL2 of thesecond arm comprises SEQ ID NO: 5; and CDRL3 of the second arm comprisesSEQ ID NO:
 10. 285. The multispecific antigen-binding construct of claim275, wherein the second arm binds PD-L1 and: a) CDRH1 of the second armcomprises SEQ ID NO: 1, CDRH2 of the second arm comprises SEQ ID NO: 11,CDRH3 of the second arm comprises SEQ ID NO: 8; CDRL1 of the second armcomprises SEQ ID NO: 9; CDRL2 of the second arm comprises SEQ ID NO: 5;and CDRL3 of the second arm comprises SEQ ID NO: 10; b) CDRH1 of thesecond arm comprises SEQ ID NO: 1, CDRH2 of the second arm comprises SEQID NO: 11, CDRH3 of the second arm comprises SEQ ID NO: 8; CDRL1 of thesecond arm comprises SEQ ID NO: 12; CDRL2 of the second arm comprisesSEQ ID NO: 5; and CDRL3 of the second arm comprises SEQ ID NO: 10; c)CDRH1 of the second arm comprises SEQ ID NO: 1, CDRH2 of the second armcomprises SEQ ID NO: 11, CDRH3 of the second arm comprises SEQ ID NO: 8;CDRL1 of the second arm comprises SEQ ID NO: 13; CDRL2 of the second armcomprises SEQ ID NO: 5; and CDRL3 of the second arm comprises SEQ ID NO:10; d) CDRH1 of the second arm comprises SEQ ID NO: 1, CDRH2 of thesecond arm comprises SEQ ID NO: 11, CDRH3 of the second arm comprisesSEQ ID NO: 8; CDRL1 of the second arm comprises SEQ ID NO: 9; CDRL2 ofthe second arm comprises SEQ ID NO: 5; and CDRL3 of the second armcomprises SEQ ID NO: 10; e) CDRH1 of the second arm comprises SEQ ID NO:1, CDRH2 of the second arm comprises SEQ ID NO: 15, CDRH3 of the secondarm comprises SEQ ID NO: 8; CDRL1 of the second arm comprises SEQ ID NO:9; CDRL2 of the second arm comprises SEQ ID NO: 5; and CDRL3 of thesecond arm comprises SEQ ID NO: 10; f) CDRH1 of the second arm comprisesSEQ ID NO: 1, CDRH2 of the second arm comprises SEQ ID NO: 16, CDRH3 ofthe second arm comprises SEQ ID NO: 17; CDRL1 of the second armcomprises SEQ ID NO: 9; CDRL2 of the second arm comprises SEQ ID NO: 5;and CDRL3 of the second arm comprises SEQ ID NO: 10; g) CDRH1 of thesecond arm comprises SEQ ID NO: 1, CDRH2 of the second arm comprises SEQID NO: 11, CDRH3 of the second arm comprises SEQ ID NO: 18, CDRL1 of thesecond arm comprises SEQ ID NO: 9; CDRL2 of the second arm comprises SEQID NO: 5; and CDRL3 of the second arm comprises SEQ ID NO: 10; h) CDRH1of the second arm comprises SEQ ID NO: 1, CDRH2 of the second armcomprises SEQ ID NO: 11, CDRH3 of the second arm comprises SEQ ID NO:19, CDRL1 of the second arm comprises SEQ ID NO: 9; CDRL2 of the secondarm comprises SEQ ID NO: 5; and CDRL3 of the second arm comprises SEQ IDNO: 10; i) CDRH1 of the second arm comprises SEQ ID NO: 1, CDRH2 of thesecond arm comprises SEQ ID NO: 11, CDRH3 of the second arm comprisesSEQ ID NO: 20, CDRL1 of the second arm comprises SEQ ID NO: 9; CDRL2 ofthe second arm comprises SEQ ID NO: 5; and CDRL3 of the second armcomprises SEQ ID NO: 10; j) CDRH1 of the second arm comprises SEQ ID NO:1, CDRH2 of the second arm comprises SEQ ID NO: 21, CDRH3 of the secondarm comprises SEQ ID NO: 22, CDRL1 of the second arm comprises SEQ IDNO: 9; CDRL2 of the second arm comprises SEQ ID NO: 5; and CDRL3 of thesecond arm comprises SEQ ID NO: 10; k) CDRH1 of the second arm comprisesSEQ ID NO: 1, CDRH2 of the second arm comprises SEQ ID NO: 29, CDRH3 ofthe second arm comprises SEQ ID NO: 8, CDRL1 of the second arm comprisesSEQ ID NO: 9; CDRL2 of the second arm comprises SEQ ID NO: 5; and CDRL3of the second arm comprises SEQ ID NO: 10; l) CDRH1 of the second armcomprises SEQ ID NO: 1, CDRH2 of the second arm comprises SEQ ID NO: 31,CDRH3 of the second arm comprises SEQ ID NO: 32, CDRL1 of the second armcomprises SEQ ID NO: 9; CDRL2 of the second arm comprises SEQ ID NO: 5;and CDRL3 of the second arm comprises SEQ ID NO: 10; m) CDRH1 of thesecond arm comprises SEQ ID NO: 1, CDRH2 of the second arm comprises SEQID NO: 33, CDRH3 of the second arm comprises SEQ ID NO: 32, CDRL1 of thesecond arm comprises SEQ ID NO: 9; CDRL2 of the second arm comprises SEQID NO: 5; and CDRL3 of the second arm comprises SEQ ID NO: 10; n) CDRH1of the second arm comprises SEQ ID NO: 1, CDRH2 of the second armcomprises SEQ ID NO: 34, CDRH3 of the second arm comprises SEQ ID NO:32, CDRL1 of the second arm comprises SEQ ID NO: 9; CDRL2 of the secondarm comprises SEQ ID NO: 5; and CDRL3 of the second arm comprises SEQ IDNO: 10; o) CDRH1 of the second arm comprises SEQ ID NO: 1, CDRH2 of thesecond arm comprises SEQ ID NO: 11, CDRH3 of the second arm comprisesSEQ ID NO: 8; CDRL1 of the second arm comprises SEQ ID NO: 9; CDRL2 ofthe second arm comprises SEQ ID NO: 5; and CDRL3 of the second armcomprises SEQ ID NO: 38; or p) CDRH1 of the second arm comprises SEQ IDNO: 1, CDRH2 of the second arm comprises SEQ ID NO: 11, CDRH3 of thesecond arm comprises SEQ ID NO: 8; CDRL1 of the second arm comprises SEQID NO: 9; CDRL2 of the second arm comprises SEQ ID NO: 5; and CDRL3 ofthe second arm comprises SEQ ID NO:
 39. 286. The multispecificantigen-binding construct of claim 275, wherein the second arm bindsPD-L1 and: a) CDRH1 of the second arm comprises SEQ ID NO: 1, CDRH2 ofthe second arm comprises SEQ ID NO: 11 and CDRH3 of the second armcomprises SEQ ID NO: 8; b) CDRH1 of the second arm comprises SEQ ID NO:1, CDRH2 of the second arm comprises SEQ ID NO: 15 and CDRH3 of thesecond arm comprises SEQ ID NO: 8; c) CDRH1 of the second arm comprisesSEQ ID NO: 1, CDRH2 of the second arm comprises SEQ ID NO: 16 and CDRH3of the second arm comprises SEQ ID NO: 17; d) CDRH1 of the second armcomprises SEQ ID NO: 1, CDRH2 of the second arm comprises SEQ ID NO: 11,and CDRH3 of the second arm comprises SEQ ID NO: 18; e) CDRH1 of thesecond arm comprises SEQ ID NO: 1, CDRH2 of the second arm comprises SEQID NO: 11, and CDRH3 of the second arm comprises SEQ ID NO: 19; f) CDRH1of the second arm comprises SEQ ID NO: 1, CDRH2 of the second armcomprises SEQ ID NO: 11, and CDRH3 of the second arm comprises SEQ IDNO: 20; g) CDRH1 of the second arm comprises SEQ ID NO: 1, CDRH2 of thesecond arm comprises SEQ ID NO: 21, and CDRH3 of the second armcomprises SEQ ID NO: 22; h) CDRH1 of the second arm comprises SEQ ID NO:1, CDRH2 of the second arm comprises SEQ ID NO: 29, and CDRH3 of thesecond arm comprises SEQ ID NO: 8; i) CDRH1 of the second arm comprisesSEQ ID NO: 1, CDRH2 of the second arm comprises SEQ ID NO: 31, and CDRH3of the second arm comprises SEQ ID NO: 32; j) CDRH1 of the second armcomprises SEQ ID NO: 1, CDRH2 of the second arm comprises SEQ ID NO: 33,and CDRH3 of the second arm comprises SEQ ID NO: 32; or k) CDRH1 of thesecond arm comprises SEQ ID NO: 1, CDRH2 of the second arm comprises SEQID NO: 34, and CDRH3 of the second arm comprises SEQ ID NO:
 32. 287. Themultispecific antigen-binding construct of claim 275, wherein the secondarm binds PD-L1 and the second arm comprises a heavy chain variableregion comprising: (i) a CDRH1 comprising SEQ ID NO: 14, (ii) a CDRH2comprising SEQ ID NO: 11 and (iii) a CDRH3 comprising SEQ ID NO: 8; (i)a CDRH1 comprising SEQ ID NO: 23, (ii) a CDRH2 comprising SEQ ID NO: 24,and (iii) a CDRH3 comprising SEQ ID NO: 8; (i) a CDRH1 comprising SEQ IDNO: 23, (ii) a CDRH2 comprising SEQ ID NO: 11, and (iii) a CDRH3comprising SEQ ID NO: 25; (i) a CDRH1 comprising SEQ ID NO: 23, (ii) aCDRH2 comprising SEQ ID NO: 26, and (iii) a CDRH3 comprising SEQ ID NO:8; (i) a CDRH1 comprising SEQ ID NO: 23, (ii) a CDRH2 comprising SEQ IDNO: 27, and (iii) a CDRH3 comprising SEQ ID NO: 28; (i) a CDRH1comprising SEQ ID NO: 23, (ii) a CDRH2 comprising SEQ ID NO: 11, and(iii) a CDRH3 comprising SEQ ID NO: 30; or (i) a CDRH1 comprising SEQ IDNO: 36, (ii) a CDRH2 comprising SEQ ID NO: 11, and (iii) a CDRH3comprising SEQ ID NO:
 37. 288. The multispecific antigen-bindingconstruct of claim 287, wherein the second arm binds PD-L1 and thesecond arm comprises a light chain variable region comprising: (i) aCDRL1 comprising SEQ ID NO: 9; (ii) a CDRL2 comprising SEQ ID NO: 5; and(iii) a CDRL3 comprising SEQ ID NO:
 10. 289. The multispecificantigen-binding construct of claim 275, wherein the second arm bindsPD-L1 and the second arm comprises a heavy chain variable regioncomprising an amino acid sequence that is at least 90% identical to anyone of SEQ ID NOs: 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, or
 58. 290. The multispecific antigen-bindingconstruct of claim 289, wherein the second arm binds PD-L1 and thesecond arm comprises a light chain variable region comprising an aminoacid sequence that is at least 90% identical to any one of SEQ ID NOs:59, 60, 61, 62, or
 63. 291. The multispecific antigen-binding constructof claim 290, wherein the second arm binds PD-L1 and the heavy chainvariable region of the second arm comprises an amino acid sequence thatis at least 90% identical to SEQ ID NO:
 55. 292. The multispecificantigen-binding construct of claim 275, wherein the construct comprisesa common light chain.
 293. The multispecific antigen-binding constructof claim 275, wherein the construct comprises at least two arms thatbind PD-1 and at least two arms that bind PD-L1.
 294. The multispecificantigen-binding construct of claim 275, wherein the construct comprisesa bivalent antibody specific for PD-1, and a bivalent antibody specificfor PD-L1.
 295. The multispecific antigen-binding construct of claim275, wherein the construct comprises a heavy chain amino acid sequencethat is at least 85% identical to the amino acid sequence of SEQ ID NO:100 or 102; and wherein the construct comprises a light chain amino acidsequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 101 or
 103. 296. The multispecific antigen-binding constructof claim 275, wherein the construct comprises a heavy chain amino acidsequence that is at least 85% identical to the amino acid sequence ofSEQ ID NO: 100, and wherein the construct comprises a light chain aminoacid sequence that is at least 85% identical to the amino acid sequenceof SEQ ID NO:
 101. 297. The multispecific antigen-binding construct ofclaim 275, wherein the construct comprises a heavy chain amino acidsequence comprising the amino acid sequence of SEQ ID NO: 100, andwherein the construct comprises a light chain amino acid sequencecomprising the amino acid sequence of SEQ ID NO:
 101. 298. A method fortreating a proliferative disorder and/or for enhancing an immuneresponse in a subject in need thereof in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a multispecific antigen-binding construct according to claim275, thereby treating the proliferative disorder in the subject. 299.The method of claim 298, wherein the proliferative disorder is cancer(e.g., a cancer selected from the group consisting of a hematologicalcancer, a neurological cancer, melanoma, breast cancer, lung cancer,head and neck cancer, a gastrointestinal cancer, liver cancer,pancreatic cancer, a genitourinary cancer, a bone cancer, and a vascularcancer).
 300. The method of claim 298, wherein the multispecificantigen-binding construct binds to PD-L1 expressed on the surface of acancer cell.
 301. An antibody or antigen-binding fragment thereof,wherein the antibody or antigen-binding fragment binds PD-1 andcomprises: (I) (a) a heavy chain variable region comprising (i) a CDRH1comprising SEQ ID NO: 70 (FTFX₁X₂YAX₃X₄, wherein X₁=S, R, G, or N; X₂=D,S, N, A, R, or G; X₃=M or L; X₄=S, L, or N); (ii) a CDRH2 comprising SEQID NO: 71; and (iii) a CDRH3 comprising SEQ ID NO: 72 (ARGLDFIVGX₅TGNDY,wherein X₅=A, Y, or R); and (b) a light chain variable regioncomprising: (i) a CDRL1 comprising SEQ ID NO: 9; (ii) a CDRL2 comprisingSEQ ID NO: 5; and (iii) a CDRL3 comprising SEQ ID NO: 10; or wherein theantibody or antigen-binding fragment binds PD-L1 and comprises: (II) (a)a heavy chain variable region comprising (i) a CDRH1 comprising SEQ IDNO: 1; (ii) a CDRH2 comprising SEQ ID NO: 2 (GGIIPX₁X₂GX₃ATYA, whereinX₁ is V or I; X₂ is F, L, or V; and X₃ is T or A); and (iii) a CDRH3comprising SEQ ID NO: 3 (ARLKX₁ELKDAFDI, wherein X₁ is G, F, or N); and(b) a light chain variable region comprising: (i) a CDRL1 comprising SEQID NO: 4 (RASQX₁ISSYLN, wherein X₁ is S, W, or Q); (ii) a CDRL2comprising SEQ ID NO: 5; and (iii) a CDRL3 comprising SEQ ID NO: 6(X₁QSYSTPLT, wherein X₁ is Q or F).
 302. The antibody or antigen-bindingfragment thereof of claim 301, wherein the antibody or antigen-bindingfragment comprises: (a) a heavy chain variable region comprising (i) aCDRH1 comprising the amino acid sequence of any one of SEQ ID NOs: 73,76, 77, 78, 79, 80, 81, 82, 83, 84, or 86; (ii) a CDRH2 comprising theamino acid sequence of SEQ ID NO: 71; and (iii) a CDRH3 comprising theamino acid sequence of any one of SEQ ID NOs: 74, 75, or 85; and (b) alight chain variable region comprising: (i) a CDRL1 comprising SEQ IDNO: 9; (ii) a CDRL2 comprising SEQ ID NO: 5; and (iii) a CDRL3comprising SEQ ID NO:
 10. 303. The antibody or antigen-binding fragmentthereof of claim 301, wherein the antibody or antigen-binding fragmentbinds PD-1 and the heavy chain variable region comprises an amino acidsequence that is at least 85% identical to the amino acid sequence ofany one of SEQ ID NOs: 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,or 99 and wherein the light chain variable region comprises an aminoacid sequence that is at least 85% identical to the amino acid sequenceof SEQ ID NO:
 59. 304. The antibody or antigen-binding fragment thereofof claim 301, wherein the antibody or antigen-binding fragment bindsPD-L1 and comprises: (a) a heavy chain variable region comprising (i) aCDRH1 comprising the amino acid sequence of any one of SEQ ID NOs: 1,14, 23 or 36; (ii) a CDRH2 comprising the amino acid sequence of any oneof SEQ ID NOs: 11, 15, 16, 21, 24, 26, 27, 29, 31, 33, or 34; and (iii)a CDRH3 comprising the amino acid sequence of any one of SEQ ID NOs: 8,17, 18, 19, 20, 22, 25, 28, 30, 32, or 37; and (b) a light chainvariable region comprising: (i) a CDRL1 comprising the amino acidsequence of any one of SEQ ID NOs: 9, 12, or 13; (ii) a CDRL2 comprisingthe amino acid sequence of SEQ ID NO: 5; and (iii) a CDRL3 comprisingthe amino acid sequence of any one of SEQ ID NOs: 10, 38 or 39.